Methods of diagnosing endometriosis

ABSTRACT

The present invention provides biomarkers for the diagnosis and prognosis of endometriosis. Generally, the methods of this invention find use in diagnosing or for providing a prognosis for endometriosis by detecting the expression levels of biomarkers, which are differentially expressed (up- or down-regulated) in endometrial cells from a patient with endometriosis. Similarly, these markers can be used to diagnose reduced fertility in a patient with endometriosis or to provide a prognosis for a fertility trial in a patient suffering from endometriosis. The present invention also provides methods of identifying a compound for treating or preventing endometriosis. Finally, the present invention provides kits for the diagnosis or prognosis of endometriosis.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/109,099, filed Apr. 24, 2008, now U.S. Pat. No. 7,871,778, whichclaims priority to provisional application, U.S. Ser. No. 60/914,018,filed Apr. 25, 2007, the contents of which are herein incorporated byreference in their entirety into this application.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made in part with Government support under grantHD031398 awarded by the National Institutes of Health. The United Statesgovernment has certain rights in this invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not Applicable

BACKGROUND OF THE INVENTION

Endometriosis is a complex disorder associated with pelvic pain andinfertility, and is characterized by the implantation of endometrialtissue outside the uterus, primarily on the pelvic peritoneum andovaries (Giudice L C, Kao L C (2004) The Lancet 364:1789-99).Endometriosis affects 6-10% of women in the general population and35-50% of women with pain and/or infertility (Eskenazi B, Warner M L(1997) Obstet Gynecol Clin North Am 24:235-58). It is widely acceptedthat by retrograde menstruation (Sampson J A (1927) Am J Obstet Gynecol14:442-469), endometrial tissue establishes itself on the peritoneum ofwomen with endometriosis due to heritable and/or acquired defects thatconfer survival advantage and promote attachment, growth,neoangiogenesis, and invasion into the peritoneum.

The main clinical symptoms of endometriosis are pelvic pain, bleedingand infertility, with the latter proposed to be related to impairedimplantation due, in part, to impaired decidualization of endometrialstromal fibroblasts (ESFs). Progesterone and cyclic AMP (cAMP) areimportant in ESF decidualization and stimulate insulin growth factorbinding protein 1 (IGFBP-1) and prolactin (PRL), markers ofdecidualization. Clinical observations suggest the presence ofprogesterone (P₄) resistance in some women with endometriosis. Inaddition, endometriotic lesions synthesize aromatase, a key enzyme inthe biosynthesis of E₂, a potential regulator of lesion growth and pain.

Though the estrogen dependence of endometriosis is well established, therole of progesterone in this disorder is comparatively less welldeveloped. The relative balance of progesterone and estrogen steroidalactivity governs the function of normal endometrium throughout themenstrual cycle. The growth promoting effects of estrogen during theproliferative phase of the cycle are countered by progesterone'santi-proliferative actions at the post-ovulatory onset of the secretoryphase and decidualizing effects on endometrial stroma later in thesecretory phase (Ferenczy A et al. (1979) Am J Obstet Gynecol133:859-67; Felix J C, Farahmand S (1997) Contraception 55:19-22). Aphenotype of attenuated progesterone response is suggested inendometriosis, and interestingly, progestin-based treatment of painassociated with this disorder is variably effective (Winkel C A, ScialliA R (2001) J Womens Health Gend Based Med 10:137-62; Metzger D A et al.(1988) Hum Pathol 19:1417-24).

The dysregulation of various progesterone target genes during theimplantation window in women with endometriosis have been reported (KaoL C et al. 2003 Endocrinology 144:2870-81; Kamat A A et al. (2004)Fertil Steril 82:1681-3; Lessey B A et al. (1994) J Clin EndocrinolMetab 79:643-9; Lessey B A et al (1992) J Clin Invest 90:188-95;Cullinan E B et al. (1996) Proc Natl Acad Sci 93:3115-20; Taylor H S etal. (1999) Hum Reprod 14:1328-31). An endometrial microenvironmentcharacterized by attenuated progesterone response may be inhospitable toembryonic implantation. Reduced responsiveness, or resistance, toprogesterone in eutopic endometrium has been implicated in thepathophysiology of this enigmatic condition, as suggested by the alteredpattern of matrix metalloproteinase (MMP) gene expression in thesecretory phase (Osteen K G et al. (2005) Fertil Steril 83:529-37).Interestingly, in vitro treatment of endometrial tissues acquired fromwomen with endometriosis with progesterone fails to fully suppresseither pro-MMP-3 or pro-MMP-7 secretion and fails to prevent the abilityof these tissues to establish experimental disease in mice (Bruner-TranK L et al. (2002) J Clin Endocrinol Metab 87:4782-91). More recently,endometrial cell culture and nude mouse models were used to demonstratethat progesterone insensitivity was intrinsic to the eutopic endometriumof women with endometriosis and could be corrected by treatment with thesynthetic progestin, tanaproget (Bruner-Tran K L et al. (2006) J ClinEndocrinol Metab 91:1554-60).

Progesterone resistance may occur at the level of the progesteronereceptor isoforms (PR-A and PR-B) (Igarashi T M et al. (2005) FertilSteril 84:67-74; Attia G R et al. (2000) J Clin Endocrinol Metab85:2897-902), steroid receptor co-activators, or down-stream effectors(TGFβ, DKK-1, Retinoic acid, c-myc, etc). In endometriotic lesions, adecrease in the expression of the progesterone target gene, 17-betahydroxysteroid dehydrogenase type I, is evidence of progesteroneresistance in ectopic endometrium (Vierikko P et al. (1985) FertilSteril 43:218-24, Bulun S E et al. (2006) Mol Cell Endocrinol248:94-103).

There is a need in the art for the identification of moleculardifferences in the endometrium of women with endometriosis in order tobetter understand the pathogenesis of this condition and to facilitatedevelopment of novel strategies for the treatment of associatedinfertility and pain. The present invention fulfills this need byidentifying biomarkers and kits useful in the diagnosis and prognosis ofendometriosis and infertility in women with endometriosis, presentingmethods for identifying compounds for treating or preventingendometriosis and infertility caused by endometriosis, and by presentingtherapeutics useful in the treatment or prevention of endometriosis andinfertility caused by endometriosis, among other embodiments.

BRIEF SUMMARY OF THE INVENTION

Generally, the methods of this invention find use in diagnosing or forproviding a prognosis for endometriosis by detecting the expressionlevels of biomarkers, which are differentially expressed (up- ordown-regulated) in endometrial cells from a patient with endometriosis.These markers can be used to distinguish the stage or severity ofendometriosis. These markers can also be used to provide a prognosis forthe course of treatment in a patient with endometriosis. Similarly,these markers can be used to diagnose infertility in a patient withendometriosis or to provide a prognosis for a fertility trial in apatient suffering from endometriosis. The biomarkers of the presentinvention can be used alone or in combination for the diagnosis orprognosis of endometriosis.

In one embodiment, the methods of the present invention find use inassigning treatment to a patient suffering from endometriosis. Bydetecting the expression levels of biomarkers found herein, theappropriate treatment can be assigned to a patient suffering fromendometriosis. These treatments can include, but are not limited to,hormone therapy, chemotherapy, immunotherapy, and surgical treatment.Similarly, the methods of the current invention can be used to assigntreatment to a patient with reduced fertility due to endometriosis. Inthis fashion, by determining the degree to which the patient's fertilityhas been reduced, through the detection of biomarkers found herein, theappropriate treatment can be assigned. Relevant treatments include, butare not limited to, hormone therapy, chemotherapy, immunotherapy, andsurgical treatment.

Diagnostic and prognostic kits comprising one or more markers for useare provided herein. Also provided by the invention are methods foridentifying compounds that are able to prevent or treat endometriosis orreduced fertility caused by endometriosis by modulating the expressionlevel or activity of markers found in any one of the identified genesubsets. Finally, therapeutic methods are provided, whereinendometriosis or reduced fertility caused by endometriosis is treatedusing antibody, siRNA, microRNA, or antisense molecules thatspecifically bind to one or more of the markers found in any one of theidentified gene subsets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. PCA of endometrium from subjects with moderate/severeendometriosis (D) and from subjects without disease (N) in the P, ES,and MS phases. Each plotted point represents an individual sample'sexpression profile distributed into a three-dimensional space based onthe variance in gene expression. The labeled axes represent three PCAcomponents and the percentage is the amount of gene expression variation(in the entire dataset) explained by each component.

FIG. 2. Hierarchial clustering analysis of endometrium from subjectswith moderate/severe endometriosis (D) and from subjects without disease(N) in the PE (red), ESE (gold) and MSE (light blue).

FIG. 3. Expression of selected genes per cycle phase in the endometriumof women with endometriosis relative to women without endometriosisusing real time PCR. A, Proliferative phase. B, Early secretory phase.C, Mid secretory phase. Each phase represents comparison of RNA samplesfrom 3 women with endometriosis and 3 women without disease. Fold-changevalues are displayed above each gene and are plotted on the y-axis on alog 10 scale. Bars represent SEM. * p<0.05, ** p<0.01.

FIG. 4. Differential expression of genes involved in the regulation ofthe mitotic cell cycle in ESE from women with versus withoutendometriosis. In this diagram, each box represents a particular gene.Up-regulated genes with fold change are represented in green whiledown-regulated genes and fold change are represented in red. Diagramadapted from KEGG at www.genome.jp.kegg.

FIG. 5A. Gene expression in severe endometriosis MSE biopsies comparedto non-endometriosis MSE samples.

FIG. 5B. Gene expression in ESFs treated with 0.5 mM cAMP for 96 hours.

FIG. 5C. Steroidogenic gene expression in ESFs treated with 0.5 mM cAMPfor 96 hours.

FIG. 5D. Steroidogenic gene expression in severe endometriosis MSEbiopsis compared to non-endometriotic MSE samples.

FIG. 5E. Gene expression in ESFs treated with E2/P4 for 14 days.

FIG. 5F. Steroidogenic gene expression in ESFs treated with E2/P4 for 14days.

FIG. 6. Graphical overview of the steroidogenesis pathway.

FIG. 7. Hic-5 protein localization during different phases of theovulatory cycle in endometrial samples taken from patients with andwithout endometriosis.

FIG. 8. Expression of Hic-5 mRNA in human endometrium throughout themenstrual cycle in patients without endometriosis.

FIG. 9. Hic-5 mRNA expression levels in endometrium from women withendometriosis relative to levels in women without endometriosisthroughout the menstrual cycle.

FIG. 10. Expression of Hic5 in human endometrial stromal fibroblastsdecidualized with 0.5 mM cAMP for 96 hours.

FIG. 11. Expression of Hic5 in human endometrial stromal fibroblastsdecidualized with 10 nM E2/1 μM P4 for 14 days.

FIG. 12. Progesterone-regulated genes in endometrial biopsies from womenwithout endometriosis.

FIG. 13. Dysregulation of progesterone-responsive genes in endometrialbiopsy from women with vs. without endometriosis.

FIG. 14. Levels of Hic-5 mRNA after transfection with control andanti-Hic-5 siRNA.

FIG. 15. Levels of Paxillin mRNA after transfection with control andanti-Hic-5 siRNA.

FIG. 16. Levels of IGFBP1, PRL, FOXO1A, and Wnt4 mRNA after transfectionwith control and anti-Hic-5 siRNA.

FIG. 17. Levels of PRA, PRB, and EBAF mRNA after transfection withcontrol and anti-Hic-5 siRNA.

FIG. 18. Levels of SST and DKK mRNA after transfection with control andanti-Hic-5 siRNA.

FIG. 19. Levels of DKK1 mRNA in non-endometriotic endometrial stromalcells transfected with siHic-5.

DETAILED DESCRIPTION OF THE INVENTION

The identification of molecular differences in the endometrium of womenwith endometriosis is an important step toward understanding thepathogenesis of this condition and toward developing novel strategiesfor the treatment of associated infertility and pain. In the presentinvention, a global gene expression analysis of endometrium from womenwith and without moderate/severe stage endometriosis was conducted.These results compared the gene expression signatures across variousphases of the menstrual cycle. Transciptome analysis revealed moleculardysregulation of the proliferative-to-secretory transition inendometrium of women with endometriosis. Paralleled gene expressionanalysis of endometrial specimens obtained during the early secretoryphase demonstrated a signature of enhanced cellular survival andpersistent expression of genes involved in DNA synthesis and cellularmitosis in the setting of endometriosis. Comparative gene expressionanalysis of progesterone-regulated genes in secretory phase endometriumconfirmed the observation of attenuated progesterone response.Additionally, susceptibility genes were identified that are associatedwith this disorder and can be used as biomarkers for diagnostic assaysand drug discovery assays, including FOXO1A, MIG6, and CYP26A1. Otherbiomarkers identified in the present invention include those found inTables 4-6 and 8-9, Hic-5, IL-8, and those found in examples 1-4.

In one embodiment, the current invention provides a method of diagnosingor providing a prognosis for endometriosis, the method comprising thestep of detecting in a biological sample altered expression (over orunder expression) of an endometriosis biomarker gene or protein in asubject suspected of or having endometriosis. In one embodiment, thegene or protein detected is selected from the group consisting of thosefound in Tables 4-6. In another embodiment, the gene or protein detectedis selected from the group consisting of those found in Tables 8 and 9.In yet another embodiment, the gene or protein detected is Hic-5, IL-8,or any other endometriosis biomarker shown to have altered expression intissues from women having endometriosis in examples 1-4.

In one embodiment of the present invention, the sample used is a biopsyfrom a mammal. In a particular embodiment, the sample is an endometrialbiopsy. In other particular embodiments, the mammal is a mouse, rabbit,horse, dog, or human. Those of skill in the art will know of othersamples well suited for use in the present invention.

In a second embodiment, the current invention provides a method ofdiagnosing or providing a prognosis for reduced fertility, the methodcomprising the step of detecting in a biological sample alteredexpression (over or under expression) of an endometriosis biomarker geneor protein in a subject suspected of or having endometriosis. In oneembodiment, the gene or protein detected is selected from the groupconsisting of those found in Tables 4-6. In another embodiment, the geneor protein detected is selected from the group consisting of those foundin Tables 8 and 9. In another embodiment, the gene or protein detectedis Hic-5, IL-8, or any other endometriosis biomarker shown to havealtered expression in tissues from women having endometriosis inexamples 1-4.

The present invention also provides methods of identifying a compoundfor treating or preventing endometriosis or reduced fertility caused byendometriosis. In one embodiment, the method comprises the steps of:contacting a compound with a protein known to be differentiallyexpressed in endometriosis and detecting altered expression as comparedto a control, thereby identifying a compound. In a particularembodiment, the protein contacted is selected from the group consistingof those found in Tables 4-6. In another embodiment, the proteincontacted is Hic-5, IL-8, any protein listed in Tables 8 and 9, or anyother protein shown to be differentially regulated in examples 1-4. Incertain embodiments, the compound is a small molecule, polynucleotide,or peptide. In other embodiments, the assay is performed in vivo, in acell, or in a tissue sample. In yet other embodiments, the assay is abiochemical assay performed iv vitro. Assays particularly well suitedfor use in the present invention are well known in the art.

The present invention also provides kits for diagnosing endometriosis orreduced fertility caused by endometriosis, comprising a probe for one ormore nucleic acid or protein biomarkers known to be differentiallyexpressed in endometriosis. In one embodiment, the biomarkers areselected from the group consisting of those in Tables 4-6. In otherembodiments, the markers are further selected from HIC-5, IL-8, proteinslisted in Tables 8 and 9, and other biomarkers shown to bedifferentially expressed herein. In one particular embodiment, the kitcomprises reagents for quantitative amplification of the selectedbiomarkers. Alternatively, the kit may comprise a microarray. In anotherparticular embodiment, the kit comprises a cocktail of antibodies. Insome embodiments the kit comprises 2 or more probes. In otherembodiments, the kits may contain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 40, 50, 75, 100, 200, 500 or more probes.

The present invention provides therapeutic molecules for the treatmentor prevention of endometriosis or reduced fertility caused byendometriosis. In one embodiment, the therapeutic molecules compriseantibodies or immunogenic fragments of antibodies. In other embodiments,the molecules comprise antisense oligonucleotides, siRNAs, microRNAs, orother nucleic acids or nucleic acid analogues well known in the art. Inparticular embodiments, the therapeutic molecules specifically hybridizeor immunogenically bind to a biomarker selected from the groupconsisting of those listed in Tables 4-6, those listed in Tables 8 and9, Hic-5, IL-8, and any other marker shown herein to be differentiallyexpressed in endometriosis. In other embodiments, the biomarkers areprogesterone-related genes.

Treatments for endometriosis are well known in the art. These treatmentsinclude, but are not limited to, pain killers, hormonal treatments,chemotherapy, and surgical treatments. Pain killers used for thetreatment of endometriosis include both simple analgesics, such asparacetamol, COX-2 inhibitors, aspirin, and other non-steroidalanti-inflammatory drugs well known in the art, and narcotic analgesics,such as morphine, codine, oxycodone, and others well known in the art.Hormonal treatments include, but are not limited to, oralcontraceptives, progestins, such as Dydrogesterone, Medroxyprogesteroneacetate, Depot medroxyprogesterone acetate, Norethisterone,Levonorgestrel, and others well known in the art, progesterone andprogesterone-like substances, GnRH agonists, such as leuprorelin,buserelin, goserelin, histrelin, deslorelin, nafarelin, and triptorelin,androgens and synthetic androgens like Danazol, and aromataseinhibitors. Surgical treatments include, but are not limited to,laparoscopic surgery, hysterectomy, and oophorectomy. Other treatmentsparticularly well suited for use in the present invention are well knownin the art.

The present invention includes other biomarkers known to bedifferentially expressed in endometriosis, such as those disclosed inBurney et al. (Burney et al., Endocrinology 148(8):3814-3826 (2007)) thecomplete contents of which are herein incorporated by reference.

DEFINITIONS

The term “marker” refers to a molecule (typically protein, nucleic acid,carbohydrate, or lipid) that is expressed in an endometrial cell from awomen with endometriosis, expressed on the surface of an endometrialcell from a woman with endometriosis, or secreted by an endometrial cellfrom a woman with endometriosis in comparison to a cell from a woman whodoes not have endometriosis, and which is useful for the diagnosis ofendometriosis, for providing a prognosis, for predicting the fertilityof an individual with endometriosis, and for preferential targeting of apharmacological agent to the endometrial cell. Oftentimes, such markersare molecules that are overexpressed in an endometrial cell from a womanwith endometriosis in comparison to a cell from a woman withoutendometriosis, for instance, 1-fold overexpression, 2-foldoverexpression, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold overexpression ormore fold-overexpression in comparison to a cell from a woman withoutendometriosis. Further, a marker can be a molecule that isinappropriately synthesized in the endometrial cell of a woman withendometriosis, for instance, a molecule that contains deletions,additions, or mutations in comparison to the molecule expressed in acell from a woman without endometriosis. Alternatively, such biomarkersare molecules that are underexpressed in an endometrial cell from awoman with endometriosis in comparison to a cell from a woman withoutendometriosis, for instance, 1-fold underexpression, 2-foldunderexpression, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold underexpression,or more fold-overexpression in comparison to a cell from a woman withoutendometriosis. Further, a marker can be a molecule that isinappropriately synthesized in a cell from a woman with endometriosis,for instance, a molecule that contains deletions, additions or mutationsin comparison to the molecule expressed in a cell from a woman withoutendometriosis.

It will be understood by the skilled artisan that markers may be used incombination with other markers or tests for any of the uses, e.g.,prediction, diagnosis, or prognosis of fertility or endometriosis,disclosed herein.

“Biological sample” includes sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histologic purposes. Suchsamples include blood and blood fractions or products (e.g., serum,plasma, platelets, red blood cells, and the like), sputum, endometrialtissue, the uterine fundus, thyroid tissue, cultured cells, e.g.,primary cultures, explants, and transformed cells, stool, urine, etc. Abiological sample is typically obtained from a eukaryotic organism, mostpreferably a mammal such as a primate e.g., chimpanzee or human; cow;dog; cat; a rodent, e.g., guinea pig, rat, Mouse; rabbit; or a bird;reptile; or fish.

A “biopsy” refers to the process of removing a tissue sample fordiagnostic or prognostic evaluation, and to the tissue specimen itself.Any biopsy technique known in the art can be applied to the diagnosticand prognostic methods of the present invention. The biopsy techniqueapplied will depend on the tissue type to be evaluated (e.g.,endometrial, etc.), the size and type of the tissue, among otherfactors. Representative biopsy techniques include, but are not limitedto, excisional biopsy, incisional biopsy, needle biopsy, surgicalbiopsy, and bone marrow biopsy. An “excisional biopsy” refers to theremoval of an entire endometrial tissue mass with a small margin ofnon-endometrial tissue surrounding it. An “incisional biopsy” refers tothe removal of a wedge of endometrial tissue. Biopsy techniques arediscussed, for example, in Harrison's Principles of Internal Medicine,Kasper, et al., eds., 16th ed., 2005, Chapter 70, and throughout Part V.

The terms “overexpress”, “overexpression”, “overexpressed”, or“up-regulated” interchangeably refer to a protein or nucleic acid (RNA)that is transcribed or translated at a detectably greater level, usuallyin an endometrial cell from a woman with endometriosis, in comparison toa cell from a woman without endometriosis. The term includesoverexpression due to transcription, post transcriptional processing,translation, post-translational processing, cellular localization (e.g.,organelle, cytoplasm, nucleus, cell surface), and RNA and proteinstability, as compared to a cell from a woman without endometriosis.Overexpression can be detected using conventional techniques fordetecting mRNA (i.e., Q-PCR, RT-PCR, PCR, hybridization) or proteins(i.e., ELISA, immunohistochemical techniques). Overexpression can be10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to acell from a woman without endometriosis. In certain instances,overexpression is 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold, or morehigher levels of transcription or translation in comparison to a cellfrom a woman without endometriosis.

The terms “underexpress”, “underexpression”, “underexpressed”, or“down-regulated” interchangeably refer to a protein or nucleic acid thatis transcribed or translated at a detectably lower level in aendometrial cell from a woman with endometriosis, in comparison to acell from a woman without endometriosis. The term includesunderexpression due to transcription, post transcriptional processing,translation, post-translational processing, cellular localization (e.g.,organelle, cytoplasm, nucleus, cell surface), and RNA and proteinstability, as compared to a control. Underexpression can be detectedusing conventional techniques for detecting mRNA (i.e., Q-PCR, RT-PCR,PCR, hybridization) or proteins (i.e., ELISA, immunohistochemicaltechniques). Underexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or less in comparison to a control. In certain instances,underexpression is 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold or morelower levels of transcription or translation in comparison to a control.

The term “differentially expressed”, “differentially regulated”, or“altered expression” refers generally to a protein or nucleic acid thatis overexpressed (upregulated) or underexpressed (downregulated) in onesample compared to at least one other sample, generally in a patientwith endometriosis, in comparison to a patient without endometriosis, inthe context of the present invention.

“Therapeutic treatment” refers to chemotherapy, hormonal therapy,radiotherapy, immunotherapy, and biologic (targeted) therapy.

By “therapeutically effective amount or dose” or “sufficient amount ordose” herein is meant a dose that produces effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site ncbi.nlm.nih.gov/BLAST or the like). Such sequencesare then said to be “substantially identical.” This definition alsorefers to, or may be applied to, the compliment of a test sequence. Thedefinition also includes sequences that have deletions and/or additions,as well as those that have substitutions. As described below, thepreferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length. The biomarkers described hereincan be detected with probes that have, e.g., more than 70% identity overa specified region, or for example, more than 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identity to the reference sequence provided by theaccession number, up to 100% identity.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1987-2005, WileyInterscience)).

A preferred example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (on the internet at www.ncbi.nlm.nih.gov/).This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses “splicevariants” and nucleic acid sequences encoding truncated forms of aprotein. Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant ortruncated form of that nucleic acid. “Splice variants,” as the namesuggests, are products of alternative splicing of a gene. Aftertranscription, an initial nucleic acid transcript may be spliced suchthat different (alternate) nucleic acid splice products encode differentpolypeptides. Mechanisms for the production of splice variants vary, butinclude alternate splicing of exons. Alternate polypeptides derived fromthe same nucleic acid by read-through transcription are also encompassedby this definition. Any products of a splicing reaction, includingrecombinant forms of the splice products, are included in thisdefinition. Nucleic acids can be truncated at the 5′ end or at the 3′end. Polypeptides can be truncated at the N-terminal end or theC-terminal end. Truncated versions of nucleic acid or polypeptidesequences can be naturally occurring or recombinantly created.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M). See, e.g., Creighton, Proteins (1984).

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al., supra.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec-2 min, an annealing phaselasting 30 sec.-2 min., and an extension phase of about 72° C. for 1-2min Protocols and guidelines for low and high stringency amplificationreactions are provided, e.g., in Innis et al. (1990) PCR Protocols, AGuide to Methods and Applications, Academic Press, Inc. N.Y.).

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding. Antibodies can bepolyclonal or monoclonal, derived from serum, a hybridoma orrecombinantly cloned, and can also be chimeric, primatized, orhumanized.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3rd ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

In one embodiment, the antibody is conjugated to an “effector” moiety.The effector moiety can be any number of molecules, including labelingmoieties such as radioactive labels or fluorescent labels, or can be atherapeutic moiety. In one aspect the antibody modulates the activity ofthe protein.

The nucleic acids of the differentially expressed genes of thisinvention or their encoded polypeptides refer to all forms of nucleicacids (e.g., gene, pre-mRNA, mRNA) or proteins, their polymorphicvariants, alleles, mutants, and interspecies homologs that (asapplicable to nucleic acid or protein): (1) have an amino acid sequencethat has greater than about 60% amino acid sequence identity, 65%, 70%,75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% or greater amino acid sequence identity, preferably over a region ofat least about 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 500, 1000, ormore amino acids, to a polypeptide encoded by a referenced nucleic acidor an amino acid sequence described herein; (2) specifically bind toantibodies, e.g., polyclonal antibodies, raised against an immunogencomprising a referenced amino acid sequence, immunogenic fragmentsthereof, and conservatively modified variants thereof; (3) specificallyhybridize under stringent hybridization conditions to a nucleic acidencoding a referenced amino acid sequence, and conservatively modifiedvariants thereof; (4) have a nucleic acid sequence that has greater thanabout 95%, preferably greater than about 96%, 97%, 98%, 99%, or highernucleotide sequence identity, preferably over a region of at least about20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 500, 1000, or morenucleotides, to a reference nucleic acid sequence. A polynucleotide orpolypeptide sequence is typically from a mammal including, but notlimited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster;cow, pig, horse, sheep, or any mammal. The nucleic acids and proteins ofthe invention include both naturally occurring or recombinant molecules.Truncated and alternatively spliced forms of these antigens are includedin the definition.

The phrase “specifically (or selectively) binds” when referring to aprotein, nucleic acid, antibody, or small molecule compound refers to abinding reaction that is determinative of the presence of the protein ornucleic acid, such as the differentially expressed genes of the presentinvention, often in a heterogeneous population of proteins or nucleicacids and other biologics. In the case of antibodies, under designatedimmunoassay conditions, a specified antibody may bind to a particularprotein at least two times the background and more typically more than10 to 100 times background. Specific binding to an antibody under suchconditions requires an antibody that is selected for its specificity fora particular protein. For example, polyclonal antibodies can be selectedto obtain only those polyclonal antibodies that are specificallyimmunoreactive with the selected antigen and not with other proteins.This selection may be achieved by subtracting out antibodies thatcross-react with other molecules. A variety of immunoassay formats maybe used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988) for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity).

The phrase “functional effects” in the context of assays for testingcompounds that modulate a marker protein includes the determination of aparameter that is indirectly or directly under the influence of abiomarker of the invention, e.g., a chemical or phenotypic. A functionaleffect therefore includes ligand binding activity, transcriptionalactivation or repression, the ability of cells to proliferate, theability to migrate, among others. “Functional effects” include in vitro,in vivo, and ex vivo activities.

By “determining the functional effect” is meant assaying for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of a biomarker of the invention, e.g., measuringphysical and chemical or phenotypic effects. Such functional effects canbe measured by any means known to those skilled in the art, e.g.,changes in spectroscopic characteristics (e.g., fluorescence,absorbance, refractive index); hydrodynamic (e.g., shape),chromatographic; or solubility properties for the protein; ligandbinding assays, e.g., binding to antibodies; measuring inducible markersor transcriptional activation of the marker; measuring changes inenzymatic activity; the ability to increase or decrease cellularproliferation, apoptosis, cell cycle arrest, measuring changes in cellsurface markers. The functional effects can be evaluated by many meansknown to those skilled in the art, e.g., microscopy for quantitative orqualitative measures of alterations in morphological features,measurement of changes in RNA or protein levels for other genesexpressed in placental tissue, measurement of RNA stability,identification of downstream or reporter gene expression (CAT,luciferase, β-gal, GFP and the like), e.g., via chemiluminescence,fluorescence, colorimetric reactions, antibody binding, induciblemarkers, etc.

“Inhibitors,” “activators,” and “modulators” of the markers are used torefer to activating, inhibitory, or modulating molecules identifiedusing in vitro and in vivo assays of endometriosis biomarkers.Inhibitors are compounds that, e.g., bind to, partially or totally blockactivity, decrease, prevent, delay activation, inactivate, desensitize,or down regulate the activity or expression of endometriosis biomarkers.“Activators” are compounds that increase, open, activate, facilitate,enhance activation, sensitize, agonize, or up regulate activity ofendometriosis biomarkers, e.g., agonists. Inhibitors, activators, ormodulators also include genetically modified versions of endometriosisbiomarkers, e.g., versions with altered activity, as well as naturallyoccurring and synthetic ligands, antagonists, agonists, antibodies,peptides, cyclic peptides, nucleic acids, antisense molecules,ribozymes, RNAi, microRNA, and siRNA molecules, small organic moleculesand the like. Such assays for inhibitors and activators include, e.g.,expressing endometriosis biomarkers in vitro, in cells, or cellextracts, applying putative modulator compounds, and then determiningthe functional effects on activity, as described above.

Samples or assays comprising endometriosis biomarkers that are treatedwith a potential activator, inhibitor, or modulator are compared tocontrol samples without the inhibitor, activator, or modulator toexamine the extent of inhibition. Control samples (untreated withinhibitors) are assigned a relative protein activity value of 100%.Inhibition of endometriosis biomarkers is achieved when the activityvalue relative to the control is about 80%, preferably 50%, morepreferably 25-0%. Activation of endometriosis biomarkers is achievedwhen the activity value relative to the control (untreated withactivators) is 110%, more preferably 150%, more preferably 200-500%(i.e., two to five fold higher relative to the control), more preferably1000-3000% higher.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, peptide,circular peptide, lipid, fatty acid, siRNA, polynucleotide,oligonucleotide, etc., to be tested for the capacity to directly orindirectly modulate endometriosis biomarkers. The test compound can bein the form of a library of test compounds, such as a combinatorial orrandomized library that provides a sufficient range of diversity. Testcompounds are optionally linked to a fusion partner, e.g., targetingcompounds, rescue compounds, dimerization compounds, stabilizingcompounds, addressable compounds, and other functional moieties.Conventionally, new chemical entities with useful properties aregenerated by identifying a test compound (called a “lead compound”) withsome desirable property or activity, e.g., inhibiting activity, creatingvariants of the lead compound, and evaluating the property and activityof those variant compounds. Often, high throughput screening (HTS)methods are employed for such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 2500 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

Predictive, Diagnostic, and Prognostic Methods

The present invention provides methods of predicting, diagnosing, orproviding prognosis of endometriosis or fertility in a patient withendometriosis by detecting the expression of markers differentiallyexpressed in cells from a patient with endometriosis. Prediction anddiagnosis involve determining the level of a panel of endometriosisbiomarker polynucleotides or the corresponding polypeptides in a patientor patient sample and then comparing the level to a baseline or range.Typically, the baseline value is representative of levels of thepolynucleotide or nucleic acid in a healthy person not suffering from,or destined to develop, endometriosis, as measured using a biologicalsample such as an endometrial biopsy or a sample of a bodily fluid.Variation of levels of a polynucleotide or corresponding polypeptides ofthe invention from the baseline range (either up or down) indicates thatthe patient has an increased risk of developing endometriosis, anincreased risk of the recurrence of endometriosis or endometrioticlesions, or an increased risk of infertility. Markers useful in thesepredictions, diagnoses, and prognoses include, but are not limited tothose found in Tables 4-6, and 8-11.

As used herein, the term “diagnosis” refers to distinguishing betweenhaving and not having endometriosis. As used herein, the term “providinga prognosis” may refer to providing a prediction of the probable courseand outcome of endometriosis or for a prediction of the probable outcomeof a treatment course for endometriosis, or alternatively for providinga prediction of the probable outcome of a fertility trial in a patientwith endometriosis.

Antibody reagents can be used in assays to detect expression levels ofthe biomarkers of the invention in patient samples using any of a numberof immunoassays known to those skilled in the art. Immunoassaytechniques and protocols are generally described in Price and Newman,“Principles and Practice of Immunoassay,” 2nd Edition, Grove'sDictionaries, 1997; and Gosling, “Immunoassays: A Practical Approach,”Oxford University Press, 2000. A variety of immunoassay techniques,including competitive and non-competitive immunoassays, can be used.See, e.g., Self et al., Curr. Opin. Biotechnol., 7:60-65 (1996). Theterm immunoassay encompasses techniques including, without limitation,enzyme immunoassays (EIA) such as enzyme multiplied immunoassaytechnique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgMantibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay(MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays(RIA); immunoradiometric assays (IRMA); fluorescence polarizationimmunoassays (FPIA); and chemiluminescence assays (CL). If desired, suchimmunoassays can be automated. Immunoassays can also be used inconjunction with laser induced fluorescence. See, e.g., Schmalzing etal., Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed.Sci., 699:463-80 (1997). Liposome immunoassays, such as flow-injectionliposome immunoassays and liposome immunosensors, are also suitable foruse in the present invention. See, e.g., Rongen et al., J. Immunol.Methods, 204:105-133 (1997). In addition, nephelometry assays, in whichthe formation of protein/antibody complexes results in increased lightscatter that is converted to a peak rate signal as a function of themarker concentration, are suitable for use in the methods of the presentinvention. Nephelometry assays are commercially available from BeckmanCoulter (Brea, Calif.; Kit #449430) and can be performed using a BehringNephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biochem.,27:261-276 (1989)).

Specific immunological binding of the antibody to nucleic acids can bedetected directly or indirectly. Direct labels include fluorescent orluminescent tags, metals, dyes, radionuclides, and the like, attached tothe antibody. An antibody labeled with iodine-125 (¹²⁵I) can be used. Achemiluminescence assay using a chemiluminescent antibody specific forthe nucleic acid is suitable for sensitive, non-radioactive detection ofprotein levels. An antibody labeled with fluorochrome is also suitable.Examples of fluorochromes include, without limitation, DAPI,fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin,R-phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labelsinclude various enzymes well known in the art, such as horseradishperoxidase (HRP), alkaline phosphatase (AP), β-galactosidase, urease,and the like. A horseradish-peroxidase detection system can be used, forexample, with the chromogenic substrate tetramethylbenzidine (TMB),which yields a soluble product in the presence of hydrogen peroxide thatis detectable at 450 nm. An alkaline phosphatase detection system can beused with the chromogenic substrate p-nitrophenyl phosphate, forexample, which yields a soluble product readily detectable at 405 nm.Similarly, a β-galactosidase detection system can be used with thechromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), whichyields a soluble product detectable at 410 nm. An urease detectionsystem can be used with a substrate such as urea-bromocresol purple(Sigma Immunochemicals; St. Louis, Mo.).

A signal from the direct or indirect label can be analyzed, for example,using a spectrophotometer to detect color from a chromogenic substrate;a radiation counter to detect radiation such as a gamma counter fordetection of ¹²⁵I; or a fluorometer to detect fluorescence in thepresence of light of a certain wavelength. For detection ofenzyme-linked antibodies, a quantitative analysis can be made using aspectrophotometer such as an EMAX Microplate Reader (Molecular Devices;Menlo Park, Calif.) in accordance with the manufacturer's instructions.If desired, the assays of the present invention can be automated orperformed robotically, and the signal from multiple samples can bedetected simultaneously.

The antibodies can be immobilized onto a variety of solid supports, suchas magnetic or chromatographic matrix particles, the surface of an assayplate (e.g., microtiter wells), pieces of a solid substrate material ormembrane (e.g., plastic, nylon, paper), and the like. An assay strip canbe prepared by coating the antibody or a plurality of antibodies in anarray on a solid support. This strip can then be dipped into the testsample and processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

Alternatively, nucleic acid binding molecules such as probes,oligonucleotides, oligonucleotide arrays, and primers can be used inassays to detect differential RNA expression in patient samples, e.g.,RT-PCR. In one embodiment, RT-PCR is used according to standard methodsknown in the art. In another embodiment, PCR assays such as Taqman®assays available from, e.g., Applied Biosystems, can be used to detectnucleic acids and variants thereof. In other embodiments, qPCR andnucleic acid microarrays can be used to detect nucleic acids. Reagentsthat bind to selected biomarkers can be prepared according to methodsknown to those of skill in the art or purchased commercially.

Analysis of nucleic acids can be achieved using routine techniques suchas Southern analysis, reverse-transcriptase polymerase chain reaction(RT-PCR), or any other methods based on hybridization to a nucleic acidsequence that is complementary to a portion of the marker codingsequence (e.g., slot blot hybridization) are also within the scope ofthe present invention. Applicable PCR amplification techniques aredescribed in, e.g., Ausubel et al. and Innis et al., supra. Generalnucleic acid hybridization methods are described in Anderson, “NucleicAcid Hybridization,” BIOS Scientific Publishers, 1999. Amplification orhybridization of a plurality of nucleic acid sequences (e.g., genomicDNA, mRNA or cDNA) can also be performed from mRNA or cDNA sequencesarranged in a microarray. Microarray methods are generally described inHardiman, “Microarrays Methods and Applications: Nuts & Bolts,” DNAPress, 2003; and Baldi et al., “DNA Microarrays and Gene Expression FromExperiments to Data Analysis and Modeling,” Cambridge University Press,2002.

Analysis of nucleic acid markers and their variants can be performedusing techniques known in the art including, without limitation,microarrays, polymerase chain reaction (PCR)-based analysis, sequenceanalysis, and electrophoretic analysis. A non-limiting example of aPCR-based analysis includes a Taqman® allelic discrimination assayavailable from Applied Biosystems. Non-limiting examples of sequenceanalysis include Maxam-Gilbert sequencing, Sanger sequencing, capillaryarray DNA sequencing, thermal cycle sequencing (Sears et al.,Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman etal., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with massspectrometry such as matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al., Nat.Biotechnol., 16:381-384 (1998)), and sequencing by hybridization. (Cheeet al., Science, 274:610-614 (1996); Drmanac et al., Science,260:1649-1652 (1993); Drmanac et al., Nat. Biotechnol., 16:54-58(1998)). Non-limiting examples of electrophoretic analysis include slabgel electrophoresis such as agarose or polyacrylamide gelelectrophoresis, capillary electrophoresis, and denaturing gradient gelelectrophoresis. Other methods for detecting nucleic acid variantsinclude, e.g., the INVADER® assay from Third Wave Technologies, Inc.,restriction fragment length polymorphism (RFLP) analysis,allele-specific oligonucleotide hybridization, a heteroduplex mobilityassay, single strand conformational polymorphism (SSCP) analysis,single-nucleotide primer extension (SNUPE) and pyrosequencing.

A detectable moiety can be used in the assays described herein. A widevariety of detectable moieties can be used, with the choice of labeldepending on the sensitivity required, ease of conjugation with theantibody, stability requirements, and available instrumentation anddisposal provisions. Suitable detectable moieties include, but are notlimited to, radionuclides, fluorescent dyes (e.g., fluorescein,fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red,tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescentmarkers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.),autoquenched fluorescent compounds that are activated bytumor-associated proteases, enzymes (e.g., luciferase, horseradishperoxidase, alkaline phosphatase, etc.), nanoparticles, biotin,digoxigenin, and the like.

Useful physical formats comprise surfaces having a plurality ofdiscrete, addressable locations for the detection of a plurality ofdifferent markers. Such formats include microarrays and certaincapillary devices. See, e.g., Ng et al., J. Cell Mol. Med., 6:329-340(2002); U.S. Pat. No. 6,019,944. In these embodiments, each discretesurface location may comprise antibodies to immobilize one or moremarkers for detection at each location. Surfaces may alternativelycomprise one or more discrete particles (e.g., microparticles ornanoparticles) immobilized at discrete locations of a surface, where themicroparticles comprise antibodies to immobilize one or more markers fordetection.

Analysis can be carried out in a variety of physical formats. Forexample, the use of microtiter plates or automation could be used tofacilitate the processing of large numbers of test samples.Alternatively, single sample formats could be developed to facilitatediagnosis or prognosis in a timely fashion.

Alternatively, the antibodies or nucleic acid probes of the inventioncan be applied to sections of patient biopsies immobilized on microscopeslides. The resulting antibody staining or in situ hybridization patterncan be visualized using any one of a variety of light or fluorescentmicroscopic methods known in the art.

In another format, the various markers of the invention also providereagents for in vivo imaging such as, for instance, the imaging oflabeled regents that detect the nucleic acids or encoded proteins of thebiomarkers of the invention. For in vivo imaging purposes, reagents thatdetect the presence of proteins encoded by endometriosis biomarkers,such as antibodies, may be labeled using an appropriate marker, such asa fluorescent marker.

Compositions, Kits and Integrated Systems

The invention provides compositions, kits and integrated systems forpracticing the assays described herein using antibodies specific for thepolypeptides or nucleic acids specific for the polynucleotides of theinvention.

Kits for carrying out the diagnostic assays of the invention typicallyinclude a probe that comprises an antibody or nucleic acid sequence thatspecifically binds to polypeptides or polynucleotides of the invention,and a label for detecting the presence of the probe. The kits mayinclude several antibodies or polynucleotide sequences encodingpolypeptides of the invention, e.g., a cocktail of antibodies thatrecognize at least two marker proteins listed in Tables 4-6 and 8-11. Inother embodiments, these cocktails may include antibodies that recognizeat least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, or moreof the marker genes listed in Tables 4-6 and 8-11.

Methods to Identify Compounds

A variety of methods may be used to identify compounds that prevent ortreat endometriosis or infertility caused by endometriosis. Typically,an assay that provides a readily measured parameter is adapted to beperformed in the wells of multi-well plates in order to facilitate thescreening of members of a library of test compounds as described herein.Thus, in one embodiment, an appropriate number of cells can be platedinto the cells of a multi-well plate, and the effect of a test compoundon the expression of a biomarker can be determined.

The compounds to be tested can be any small chemical compound, or amacromolecule, such as a protein, sugar, nucleic acid or lipid.Typically, test compounds will be small chemical molecules and peptides.Essentially any chemical compound can be used as a test compound in thisaspect of the invention, although most often compounds that can bedissolved in aqueous or organic (especially DMSO-based) solutions areused. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one embodiment, high throughput screening methods are used whichinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds. Such “combinatorialchemical libraries” or “ligand libraries” are then screened in one ormore assays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. In this instance, such compounds are screenedfor their ability to reduce or increase the expression of the biomarkersof the invention.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries are wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res., 37:487-493(1991) and Houghton et al., Nature, 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., PNASUSA, 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J.Amer. Chem. Soc., 114:6568 (1992)), nonpeptidal peptidomimetics withglucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc.,114:9217-9218 (1992)), analogous organic syntheses of small compoundlibraries (Chen et al., J. Amer. Chem. Soc., 116:2661 (1994)),oligocarbamates (Cho et al., Science, 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem., 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see; e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, 5,288,514, and thelike).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.)

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 96 modulators. If 1536 well plates are used, thena single plate can easily assay from about 100—about 1500 differentcompounds. It is possible to assay many plates per day; assay screensfor up to about 6,000, 20,000, 50,000, or 100,000 or more differentcompounds is possible using the integrated systems of the invention.

Methods to Inhibit Marker Protein Expression Using Nucleic Acids

A variety of nucleic acids, such as antisense nucleic acids, siRNAs,microRNAs, or ribozymes, may be used to inhibit the function of themarkers of this invention. Ribozymes that cleave mRNA at site-specificrecognition sequences can be used to destroy target mRNAs, particularlythrough the use of hammerhead ribozymes. Hammerhead ribozymes cleavemRNAs at locations dictated by flanking regions that form complementarybase pairs with the target mRNA. Preferably, the target mRNA has thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art.

Gene targeting ribozymes necessarily contain a hybridizing regioncomplementary to two regions, each of at least 5 and preferably each 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguousnucleotides in length of a target mRNA. In addition, ribozymes possesshighly specific endoribonuclease activity, which autocatalyticallycleaves the target sense mRNA.

With regard to antisense, siRNA, microRNAs, or ribozymeoligonucleotides, phosphorothioate oligonucleotides can be used.Modifications of the phosphodiester linkage as well as of theheterocycle or the sugar may provide an increase in efficiency.Phophorothioate is used to modify the phosphodiester linkage. An N3′-P5′phosphoramidate linkage has been described as stabilizingoligonucleotides to nucleases and increasing the binding to RNA. Peptidenucleic acid (PNA) linkage is a complete replacement of the ribose andphosphodiester backbone and is stable to nucleases, increases thebinding affinity to RNA, and does not allow cleavage by RNAse H. Itsbasic structure is also amenable to modifications that may allow itsoptimization as an antisense component. With respect to modifications ofthe heterocycle, certain heterocycle modifications have proven toaugment antisense effects without interfering with RNAse H activity. Anexample of such modification is C-5 thiazole modification. Finally,modification of the sugar may also be considered. 2′-O-propyl and2′-methoxyethoxy ribose modifications stabilize oligonucleotides tonucleases in cell culture and in vivo.

“RNAi molecule” or an “siRNA” refers to a nucleic acid that forms adouble stranded RNA, which double stranded RNA has the ability to reduceor inhibit expression of a gene or target gene when the siRNA expressedin the same cell as the gene or target gene. “siRNA” thus refers to thedouble stranded RNA formed by the complementary strands. Thecomplementary portions of the siRNA that hybridize to form the doublestranded molecule typically have substantial or complete identity. Inone embodiment, an siRNA refers to a nucleic acid that has substantialor complete identity to a target gene and forms a double stranded siRNA.The sequence of the siRNA can correspond to the full length target gene,or a subsequence thereof. Typically, the siRNA is at least about 15-50nucleotides in length (e.g., each complementary sequence of the doublestranded siRNA is 15-50 nucleotides in length, and the double strandedsiRNA is about 15-50 base pairs in length, preferable about preferablyabout 20-30 base nucleotides, preferably about 20-25 nucleotides inlength, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotidesin length.

Inhibitory oligonucleotides can be delivered to a cell by directtransfection or transfection and expression via an expression vector.Appropriate expression vectors include mammalian expression vectors andviral vectors, into which has been cloned an inhibitory oligonucleotidewith the appropriate regulatory sequences including a promoter to resultin expression of the antisense RNA in a host cell. Suitable promoterscan be constitutive or development-specific promoters. Transfectiondelivery can be achieved by liposomal transfection reagents, known inthe art (e.g., Xtreme transfection reagent, Roche, Alameda, Calif.;Lipofectamine formulations, Invitrogen, Carlsbad, Calif.). Deliverymediated by cationic liposomes, by retroviral vectors and directdelivery are efficient. Another possible delivery mode is targetingusing antibodies to cell surface markers for the target cells.

For transfection, a composition comprising one or more nucleic acidmolecules (within or without vectors) can comprise a delivery vehicle,including liposomes, for administration to a subject, carriers anddiluents and their salts, and/or can be present in pharmaceuticallyacceptable formulations. Methods for the delivery of nucleic acidmolecules are described, for example, in Gilmore, et al., Curr DrugDelivery (2006) 3:147-5 and Patil, et al., AAPS Journal (2005)7:E61-E77, each of which are incorporated herein by reference. Deliveryof siRNA molecules is also described in several U.S. PatentPublications, including for example, 2006/0019912; 2006/0014289;2005/0239687; 2005/0222064; and 2004/0204377, the disclosures of each ofwhich are hereby incorporated herein by reference. Nucleic acidmolecules can be administered to cells by a variety of methods known tothose of skill in the art, including, but not restricted to,encapsulation in liposomes, by iontophoresis, by electroporation, or byincorporation into other vehicles, including biodegradable polymers,hydrogels, cyclodextrins (see, for example Gonzalez et al., 1999,Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCTpublication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. 2002/130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In another embodiment,the nucleic acid molecules of the invention can also be formulated orcomplexed with polyethyleneimine and derivatives thereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives.

Examples of liposomal transfection reagents of use with this inventioninclude, for example: CellFectin, 1:1.5 (M/M) liposome formulation ofthe cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine anddioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); Cytofectin GSV,2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); DOTAP(N41-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); Lipofectamine, 3:1 (M/M) liposome formulation ofthe polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL); and(5) siPORT (Ambion); HiPerfect (Qiagen); X-treme GENE (Roche);RNAicarrier (Epoch Biolabs) and TransPass (New England Biolabs).

In some embodiments, antisense, siRNA, microRNAs, or ribozyme sequencesare delivered into the cell via a mammalian expression vector. Forexample, mammalian expression vectors suitable for siRNA expression arecommercially available, for example, from Ambion (e.g., pSilencervectors), Austin, Tex.; Promega (e.g., GeneClip, siSTRIKE, SiLentGene),Madison, Wis.; Invitrogen, Carlsbad, Calif.; InvivoGen, San Diego,Calif.; and Imgenex, San Diego, Calif. Typically, expression vectors fortranscribing siRNA molecules will have a U6 promoter.

In some embodiments, antisense, siRNA, microRNAs, or ribozyme sequencesare delivered into cells via a viral expression vector. Viral vectorssuitable for delivering such molecules to cells include adenoviralvectors, adeno-associated vectors, and retroviral vectors (includinglentiviral vectors). For example, viral vectors developed for deliveringand expressing siRNA oligonucleotides are commercially available from,for example, GeneDetect, Bradenton, Fla.; Ambion, Austin, Tex.;Invitrogen, Carlsbad, Calif.; Open BioSystems, Huntsville, Ala.; andImgenex, San Diego, Calif.

EXAMPLES Example 1

In this example, paralleled gene expression analysis was applied toinvestigate cycle phase-dependent differences in the eutopic endometrialgene expression signatures across the menstrual cycle of women withmoderate/severe disease, compared to women without endometriosis. Inwomen with moderate/severe disease, the gene expression profilesuggested incomplete transitioning of the endometrium from theproliferative to early secretory phase, a phenotype of enhanced cellularsurvival, and attenuation of progesterone-induced down-regulation of DNAsynthesis and cellular mitosis. Additionally, the secretory endometriumfrom women with disease demonstrated dysregulation of numerous genesknown to be progesterone regulated. These results provide compellingmolecular evidence for attenuated progesterone responsiveness withineutopic endometrium in women with endometriosis.

Endometriosis is a visually heterogeneous condition and studies havedocumented inaccuracy in its visual diagnosis, particularly in cases ofminimal/mild stages (Marchino G L et al. (2005) Fertil Steril 84:12-5;Walter A J et al. (2001) Am J Obstet Gynecol 184:1407-11; discussion1411-3). The present study avoided these problems by including onlywomen with surgically documented and histologically validatedmoderate/severe stage endometriosis. Accordingly, endometrial biopsieswere obtained from normally cycling women with histologically confirmed,moderate-severe endometriosis at laparoscopy (n=21) and from normallycycling women found to be free of endometriosis at surgery (n=16).Moderate to severe endometriosis (Stage disease) was defined inaccordance with the Revised American Fertility Society (rAFS)classification system (The American Fertility Society (1985) FertilSteril 43:351-2). Study subjects in the severe endometriosis cohort were22-44 years old, had regular menstrual cycles, and were documented notto be pregnant at the time of surgery. Many of these patients were alsoinfertile and several had failed in vitro fertilization treatment(s)prior to laparoscopic surgery (Littman E et al. (2005) Fertil Steril84:1574-8). The demographic profile of the endometriosis-free cohort hasbeen described previously (Talbi S et al. (2006) Endocrinology147:1097-121). The demographic profile of the cohort with endometriosisis provided in Table 1. Subjects using any form of hormonal treatmentwithin 3 months of biopsy were excluded from the study. Biopsy specimenswere obtained using either Pipelle catheters or curette from the uterinefundus under sterile conditions. In comparable subjects withoutendometriosis, we have reported minimal variation between these samplingmethods when comparing endometrial molecular profiles (Talbi S et al.(2006) Endocrinology 147:1097-121). Samples were processed forhistologic confirmation as well as for RNA isolation. Endometrium wasdated by up to four independent histopathologists, all of whom wereblinded to the subject's identity and timing of the biopsy. Histologicdating was based upon the method of Noyes et al (Noyes R W et al. (1975)Am J Obstet Gynecol 122:262-3). Specimens were classified asproliferative (PE, days 8-14), early secretory (ESE, days 15-18),mid-secretory (MSE, days 19-23) or late secretory (LSE, days 24-28)endometrium.

TABLE 1 Subject characteristics - moderate/severe endometriosis cohort(n = 21) Patient Cycle Age Distri- ID phase (yr) bution DiagnosesEthnicity Medications 26A Pro 31 O, PI Caucasian 587 Pro 37 PI Liverendo Caucasian 647 Pro 39 R, O, PI Leiomyoma Caucasian 594 Pro 38 O, PICaucasian Amour thyroid 651 Pro 37 O, PI Leiomyoma Caucasian Advair,rhinocort 508 Pro 25 R, O, PI Caucasian Atenolol 489 ES 39 R, PILeiomyoma Asian Levothy- roxine 496 ES 37 O, PI Leiomyoma CaucasianAdvair, rhinocort 599 ES 35 R, O, PI Black 27A ES 22 R, O, PI Caucasian517 ES 35 R, PI Leiomyoma Asian Trental, ciprofloxacin 575 ES 26 O, PIUnknown 33A MS 27 R, PI Caucasian 7A/97A MS 35 O, PI Unknown 73A MS 26O, PI Caucasian 516 MS 34 R, PI Leiomyoma Asian 540 MS 37 R, PICaucasian Keflex prn 543 MS 38 R, O, PI Caucasian 678 MS 44 R, PILeiomyoma Asian 72A MS 31 PI Caucasian 645 MS 39 R, PI Asian IndianUnder the Cycle phase, Pro = proliferative; ES = early secretory; MS =mid secretory. Under the Distribution of disease, PI = peritonealendometriosis, defined as biopsy proven serosal implant; O = ovarianendometriosis, defined as biopsy proven endometrioma; R = rectovaginalendometriosis, defined as posterior cul de sac obliteration due toendometriotic lesions. All endometrial specimens were taken fromsubjects surgically staged with moderate/severe endometriosis inaccordance with rAFS criteria (23).

RNA Preparation/Target Preparation/Array Hybridization and Scanning

A total of 37 specimens were used for microarray analysis, with 21specimens (PE=6, ESE=6, MSE=9) obtained from subjects surgicallyconfirmed to be affected by moderate-severe endometriosis and 16specimens (PE=5, ESE=3, MSE=8) obtained from subjects surgicallyconfirmed to be free of endometriosis. The latter samples were usedpreviously to define the normal endometrial expression signature acrossthe various phases of the menstrual cycle (Talbi S et al. (2006)Endocrinology 147:1097-121). Each endometrial biopsy specimen wasprocessed individually for microarray hybridization. Briefly, total RNAwas extracted from each whole tissue specimen using Trizol Reagent(Invitrogen, Carlsbad, Calif.), subjected to DNase treatment andpurified using the RNeasy Kit (Qiagen, Valencia, Calif.). RNA qualitywas confirmed by A260/A280 ratio and agarose gel electrophoresis, whereresolution of distinct 28s and 18s rRNA bands was used to suppose intactRNA. Using 5 μg of template, double stranded cDNA and biotinylated cRNAwere prepared by methods previously described (Talbi S et al. (2006)Endocrinology 147:1097-121). After chemical fragmentation with 5×fragmentation buffer (200 mM Tris, pH 8.1; 500 mM KOAc; 150 mM MgOAc),biotinylated cRNAs were hybridized to Affymetrix HU133 Plus 2.0 versionhigh density oligonucleotide arrays (Affymetrix, Santa Clara, Calif.) onan Affymetrix fluidics station at the Stanford University School ofMedicine Protein and Nucleic Acid (PAN) Facility. Fluorescent labelingof samples and laser confocal scanning of the arrays were conducted atthe PAN facility.

Microarray Gene Expression Data Analysis

The data generated by the Affymetrix GeneChip Operating Softwareanalysis of the scanned array images were imported into GeneSpringversion 7.2 (Agilent Technologies Inc., Santa Clara, Calif.) foranalysis. The data files containing the probe level intensities wereprocessed using the Robust Microarray Analysis (RMA) algorithm(GeneSpring) for background adjustment, normalization and log2-transformation of perfect match values (Kamat AA et al. (2004) FertilSteril 82:1681-3). Per-chip and per-gene normalization was conductedusing GeneSpring normalization algorithms. The normalized data were usedin pairwise comparisons of cycle phase-specific endometrium fromsubjects with and without moderate-severe endometriosis. The resultinggene lists from each pairwise comparison included only the genes thatevidenced a fold change of 1.5 or higher and a p-value less than 0.05 bya one-way ANOVA parametric test and a Benjamini-Hochberg multipletesting correction for false discovery rate, as described (Talbi S etal. (2006) Endocrinology 147:1097-121). To identify samples with similarpatterns of gene expression, principal component analysis (PCA) wasperformed in which a multi-dimensional dataset is displayed in reduceddimensionality, with each dimension representing a component to which acertain percentage of variance in the data is attributed. The PCAalgorithm in GeneSpring was applied to all endometrial specimens groupedby disease status and cycle phase using all 54,600 genes and ESTs on theHG U133 Plus 2.0 chip to evaluate for similar gene expression patternsand underlying cluster structures, as described (Talbi S et al (2006)Endocrinology 147:1097-121). To further evaluate for patterns in thegene expression profiles, hierarchial clustering analysis of thecombined (pairwise comparisons-derived) gene list and all samples wasconducted using the smooth correlation for distance measure algorithm(GeneSpring). A Heatmap was generated which graphically depicts themeasured intensity values of the genes and the dendrogram illustratesrelationships between the specimens (Talbi S et al. (2006) Endocrinology147:1097-121). Raw data files of this experiment are stored at the NCBIGene Expression Omnibus (GEO) database under the identifier GSE6364.

Gene Ontology Classification of Differentially Expressed Genes.

The integration of gene expression data with the gene ontology wascarried out using the GO Tree Machine (GOTM) (Ashburner M et al. (2000)Nat Genet 25:25-9). GOTM builds significant biological processes,molecular functions, and cellular components in a gene list aspreviously described (Osteen K G et al. (2005) Fertil Steril 83:529-37).

Validation of Microarray Data by Real-Time PCR.

Genes of different expression fold changes in each menstrual cycle phasewere selected for validation by real time PCR as described previously(Talbi S et al. (2006) Endocrinology 147:1097-121). Real time PCR wasperformed on a minimum of N=3 samples in both the normal and diseaseconditions for the proliferative, early-secretory, and mid-secretoryphases. First strand cDNA was generated from 1 μg of total RNA using theOmniscript RT Kit (Qiagen, Valencia, Calif.). PCR reactions wereperformed in triplicate in 25 μL using the Brilliant SYBR Green PCR kitaccording to the manufacturer's (Stratagene) specifications. Ribosomalprotein L19 (RPL19) was chosen for use as a normalizer due to the lowvariation in expression levels evidenced by this gene in the microarraydataset. Intron spanning PCR primers were designed for each gene ofinterest (Table 2). Data analysis of the real-time PCR data wasconducted as described previously (Talbi S et al. (2006) Endocrinology147:1097-121). We considered the normal endometrial specimens as“control” samples, and the endometrial specimens from subjects withsevere endometriosis as our “test” samples when conducting fold changecalculations from the raw Ct values. Statistical analysis of the PCRdata was conducted using the relative expression software tool (REST)algorithm, which employs a pairwise fixed reallocation and randomizationtest to determine significance (Pfaffl M W et al. (2002) Nucleic AcidsRes 30:e36).

TABLE 2 Primer sequences used in real-time PCR reactions. ExpectedProduct Gene Forward Primer (5′-3′) Reverse primer (5′-3′) Unigene IDLength S100A8 CAGCTGTCTTTCAGAAGACCTG TGAGGACACTCGGTCTCTAGC Hs.416073153 bp SEQ ID NO: 1 SEQ ID NO: 2 SUI1 ATTGAGCATCCGGAATATGGTGATCGTCCTTAGCCAGTCC Hs.150580 101 bp SEQ ID NO: 3 SEQ ID NO: 4 LTFGACTCCATGGCAAAACAACA GAGGAATTCACAGGCTTCCA Hs.529517 121 bp SEQ ID NO: 5SEQ ID NO: 6 IHH CGGCTTTGACTGGGTGTATT GAAAATGAGCACATCGCTGA Hs.654504217 bp SEQ ID NO: 7 SEQ ID NO: 8 Patched TCGAAGGTGGAAGTCATTGAGCACAGGGCATCTTTTCCATAA Hs.494538 184 bp SEQ ID NO: 9 SEQ ID NO: 10 OVGP1TATGTCCCGTATGCCAACAA ACGTAGACAAGGGGGAAAGG Hs.1154 253 bp SEQ ID NO: 11SEQ ID NO: 12 TOP2A AAGCCCTCCTGCTACACATTT CAGGCTTTTGAGAGACACCAGHs.156346 191 bp SEQ ID NO: 13 SEQ ID NO: 14 CDK1 GCTTATGCAGGATTCCAGGTTCAATCCCCTGTAGGATTTGGT Hs.334562 143 bp SEQ ID NO: 15 SEQ ID NO: 16FLJ10540 CTCAAGACCGTTGTCTCTTCG TTCCCACTTGTGATTTCATCC Hs.14559 197 bpSEQ ID NO: 17 SEQ ID NO: 18 MT1H GCAAGTGCAAAAAGTGCAAATCACTTCTCTGACGCCCCTTT Hs.438462 115 bp SEQ ID NO: 19 SEQ ID NO: 20SCGB2A2 ACCATGAAGTTGCTGATGGTC GGCATTTGTAGTGGCATTGTC Hs.46452 177 bpSEQ ID NO: 21 SEQ ID NO: 22 CYP26A1 GCATCGAGCAGAACATTCGTGGAGAACATGTGGGTAGAGC Hs.150595 235 bp SEQ ID NO: 23 SEQ ID NO: 24 PSDAGCTCCCAAAAGAAGTTCAGC ACTCCAGGTAGGCCTCCTTCT Hs.154658 199 bpSEQ ID NO: 25 SEQ ID NO: 26 SH3D5 CCACAGAATGATGATGAGTTGGGTTGCCTGGAAAAGTACCAAA Hs.696027 126 bp SEQ ID NO: 27 SEQ ID NO: 28 TACC2AGGAGAGCCCTGTCAAGTCAT CTTCTGGGAGGATTTCTCTGG Hs.501252 185 bpSEQ ID NO: 29 SEQ ID NO: 30 SEMA3C AAGTCTCCGCAGGCATCTATCCAACAGCCACCATTTCTGAAT Hs.269109 226 bp SEQ ID NO: 31 SEQ ID NO: 32 BIRC5CACTGAGAACGAGCCAGACTT AACCGGACGAATGCTTTTTAT Hs.514527 110 bpSEQ ID NO: 33 SEQ ID NO: 34 ERRFI1/MIG6 TTGCTGCTCAGGAGATCAGATTCAGACTGTAGGCCATGGTT Hs.605445 154 bp SEQ ID NO: 35 SEQ ID NO: 36 ERBB2CCCTGGTCACCTACAACACAG CTCTGCTGTCACCTCTTGGTT Hs.446352 167 bpSEQ ID NO: 37 SEQ ID NO: 38 FOXO1 AAGAGCGTGCCCTACTTCAACTGTTGTTGTCCATGGATGC Hs.370666 209 bp SEQ ID NO: 39 SEQ ID NO: 40 PLZFCCACCCCTACGAGTGTGAGT GCTTGATCATGGCCGAGTAG Hs.591945 230 bp SEQ ID NO: 41SEQ ID NO: 42 SPP1 AGAAGTTTCGCAGACCTGACA GTCATCCAGCTGACTCGTTTC Hs.313182 bp SEQ ID NO: 43 SEQ ID NO: 44

Cluster Analysis.

Principal component analysis (PCA) revealed that endometrial samplesfrom subjects with endometriosis cluster by cycle phase with samplesfrom subjects without disease (FIG. 1). PCA depicts the variance in geneexpression profiles among specimens. For purposes of comparison, sampleswere grouped by cycle phase. On the three dimensional graphic, thedistance between two plotted points is proportional to the degree ofsimilarity between the two groups' gene expression profiles, using allof the genes and ESTs on the Affymetrix gene chip HG 133 Plus 2.0.Clustering was more dependent on cycle phase than endometriosis status.The largest variance between specimens from subjects with and withoutmoderate/severe endometriosis was observed in the early secretory phase.Interestingly, the ESE specimens from women with endometriosiscollectively plotted much closer to the PE specimens than did the normalESE specimens, suggestive of attenuation of the progesterone mediatedtransition on the molecular level.

Unsupervised hierarchial clustering analysis was conducted using thegene expression profiles of the 37 endometrial samples (21 withendometriosis, 16 without endometriosis) based on the combined list ofgenes showing differential expression throughout the comparable phasesof the menstrual cycle. As evidenced by the dendrogram of sampleclustering (FIG. 2), the samples self-segregate according to cyclephase, confirming our previously reported observation of phase dependentsegregation of endometrial samples (Talbi S et al. (2006) Endocrinology147:1097-121). Additionally, within the early secretory cycle phase, thesamples demonstrate striking self-segregation into normal and diseaseclusters. Three endometrial specimens sampled from patients withendometriosis (599, 517, and 27A) were classified as late proliferativephase by the Noyes criteria (Noyes RW et al. (1975) Am J Obstet Gynecol122:262-3), mostly on the basis of an increased number of mitoticfigures observed in these histologic preparations. However, eachspecimen's overall gene expression profile clustered with the earlysecretory phase specimens. Dating of these specimens based on lastmenstrual period placed them collectively between cycle days 15 and 17,confirming their molecular-based dating in the early secretory phase. Tofurther clarify the dating of these specimens, microarray analysis wasconducted comparing these three specimens with the other three ESEspecimens (489, 496, and 575). This sub-analysis showed no significantdifferences, thereby validating their correct classification as earlysecretory.

Expression profiling reveals persistent expression of genes involved incellular proliferation in ESE from women with endometriosis.

Of the three phases of the menstrual cycle investigated, the earlysecretory phase involved the greatest number of statisticallysignificant and differentially expressed genes in endometrium from womenwith versus without endometriosis (Table 3). The most highly up- anddown-regulated genes are shown in Table 4. The complete gene lists forall cycle phases in women with disease versus women withoutendometriosis are provided in Burney et al. (Burney et al.,Endocrinology 148(8):3814-3826 (2007)). The data have been submitted tothe GEO database under the identifier GSE6364. The gene ontologies (GOs)enriched in the ESE of women with endometriosis are mostly involved withmitosis and cell proliferation (Table 7), processes which, in womenwithout disease, are normally down-regulated in ESE (and up-regulated inPE). The complete GO categories for all phases are provided in (Burneyet al., Endocrinology 148(8):3814-3826 (2007)).

TABLE 3 Number of significantly differentially expressed genes inendometrium of endometriosis versus normalsubjects at indicated foldchange thresholds. Fold Change 1.5x 2.0x 4.0x Menstrual phase Up Down UpDown Up Down Proliferative 252 447 24 14 2 0 Early secretory 747 1741213 521 26 59 Mid secretory 428 293 4 22 0 0

TABLE 4 Most highly up- and down-regulated genes per cyclephase-dependent comparison. Fold changes provided compare theendometrium from women with vs. without moderate/severe endometriosis.Unigene Fold Gene symbol Description ID Change p value ProliferativePhase Up regulated S100A8 S100 calcium binding protein A8 Hs.416073 4.950.0111 SUI1 Putative translation initiation factor Hs.150580 3.74 0.0136LTF Lactotransferrin Hs.529517 3.51 0.0357 GRAP GRB2-related adaptorprotein Hs.567416 2.9 0.0125 CD163 CD163 antigen Hs.504641 2.63 0.0038Down regulated DEAD/H DEAD (Asp-Glu-Ala-Asp) SEQ ID NO: 45 box Hs.528305−2.63 0.0015 polypeptide ORM2 Orosomucoid 2 Hs.522356 −2.56 0.0383 PGRProgesterone receptor AI378893 −2.33 0.0235 Hs.32405 IHH Indian hedgehoghomolog Hs.654504 −2.33 0.029 OVGP1 Oviductal glycoprotein 1 precursorHs.1154 −2.32 0.0362 Early Secretory Phase Up regulated TOP2ATopoisomerase (DNA) II alpha 170 kDa Hs.156346 7.59 0.0003 RAB6KIFL RAB6interacting, kinesin-like Hs.73625 5.19 0.0014 Apolipoprotein B mRNAediting enzyme, APOBEC3B catalytic polypeptide-like 3B Hs.226307 5.120.0011 PENK Proenkephalin Hs.339831 5.08 <0.05 TOPK T-LAKcell-originated protein kinase Hs.104741 5.05 0.0021 Down regulated MT1YMetallothionein 1Y Hs.647370 −11.1 0.0079 CXCL13: Chemokine ligand 13(B-cell SCYB13 chemoattractant) Hs.100431 −9.09 0.0018 CYP26A1Cytochrome P450, subfamily XXVIA Hs.150595 −8.33 0.0306 SCGB2A2Secretoglobin, family 2A, member 2 Hs.46452 −7.69 0.0203 MT1GMetallothionein 1G Hs.433391 −7.14 0.0054 Mid Secretory Phase Upregulated S100A8 S100 calcium binding protein A8 Hs.416073 2.11 0.0156BLTR2 Leukotriene B4 receptor BLTR2 Hs.642693 2.02 0.0005 MAPK4Mitogen-activated protein kinase 4 Hs.433728 1.98 0.00001 PTAFRPlatelet-activating factor receptor Hs.433540 1.91 0.002 GZMA Granzyme AHs.90708 1.87 0.0286 Down regulated SCGB2A2 Secretoglobin, family 2A,member 2 Hs.46452 −3.33 0.0447 MMP26 Matrix metalloproteinase 26Hs.204732 −3.03 0.0457 CYP26A1 Cytochrome P450, subfamily XXVIAHs.150595 −2.63 0.0306 POMZP3 POM (POM121 homolog, rat) and ZP3 fusionHs.488877 −2.56 0.0019 DEPP/C10orf10 Chromosome 10 open reading frame 10Hs.93675 −2.38 0.0129

To further define the observation of a persistent cellular proliferationsignature in early secretory phase endometrium from women withendometriosis, we examined expression of individual genes mapped in theKEGG cell cycle pathway (FIG. 4, for KEGG pathway analysis for allphases). As demonstrated, multiple genes involved in mitotic cell cycleregulation are differentially expressed. The finding of a coherentpattern of consistent dysregulation among multiple genes involved in apathway or process improves the robustness of the finding. Importantly,gene ontology analysis of differentially expressed genes in theproliferative phase did not demonstrate enrichment for genes involved inthe cell cycle in endometrium from women with endometriosis. Therefore,the finding of a proliferative gene expression profile persisting inearly secretory endometrium of these women is consistent with reducedprogesterone mediated inhibition of estrogen-induced cellular mitosis.

Progesterone Regulated Genes in ESE and MSE from Women with Versuswithout Endometriosis.

Genes known to be progesterone-regulated for dysregulation during thesecretory phase in the endometrium from women with endometriosis werefurther investigated. Progesterone-regulated genes were identified bysystematic review of the literature using the PubMed search engine, andwere compared against our dataset of differentially expressed genes inthe secretory phases among women with versus without endometriosis. Thisapproach revealed fifty-four and sixteen dysregulated genes in the ESEand MSE, respectively (Table 5).

TABLE 5 Genes previously shown to be progesterone regulated that aredysregulated in endometrium of subjects with moderate/severeendometriosis. A, Early secretory phase. B, Mid secretory phase. Valuesindicate fold change of each gene in eutopic endometrium from subjectswith endometriosis relative to control endometrium. Evidence for P- Foldp Gene symbol Description regulation Unigene ID change value A. Earlysecretory phase (n = 54) Down regulated MT1Y Metallothionein 1Y (25)Hs.647370 −11.1 0.0079 CYP26A1 Cytochrome P450, subfamily (54) Hs.150595−8.33 0.0306 XXVIA SCGB2A2 Secretoglobin, family 2A, member 2 (25, 73)Hs.46452 −7.69 0.0203 MT1G Metallothionein 1G (25, 73, 74) Hs.433391−7.14 0.0054 MT1X Metallothionein 1X (25, 75) Hs.374950 −7.14 0.0046MT1F Metallothionein 1F (25) Hs.513626 −6.67 0.0073 CAPN6 Calpain 6 (25,54) Hs.496593 −6.25 0.0018 MT1H Metallothionein 1H (25) Hs.438462 −5.560.0092 SPP1 Secreted phosphoprotein 1, (25, 74) Hs.313 −5.56 0.0154osteopontin) MT1E Metallothionein 2A (25, 73-75) Hs.647371 −4.55 0.0157ISG20 Interferon stimulated gene, 20-Kd (54) Hs.459265 −3.85 0.0008 MAOAMonoamine oxidase A (25, 74, 76) Hs.183109 −3.03 0.0022 GPX3 Glutathioneperoxidase 3 (74) Hs.386793 −2.78 0.0172 MIG6/ERRFI1 Mitogen-induciblegene 6 (77) Hs.605445 −2.7 0.0025 SGK Serum/glucocorticoid regulated(54, 75) Hs.725237 −2.63 0.0296 kinase DEPP/C10orf10 Chromosome 10 openreading (75, 78) Hs.93675 −2.5 0.0234 frame 10 DKK1 Dickkopf homolog 1(8, 25, 74, Hs.40499 −2.44 <0.05 75, 79) MUC1 Mucin 1, transmembrane(25) Hs.89603 −2.38 0.0094 PAEP Progestagen-associated (80-82) Hs.532325−2.33 <0.05 endometrial protein/Glycodelin FOXO1A Forkhead box O1A (25,83) Hs.370666 −2.27 0.016 STC1 Stanniocalcin 1 (84) Hs.25590 −2.080.0476 BCAT1 Branched-chain aminotransferase 1 (54) Hs.438993 −2 0.0467BCL6 B-cell CLL/lymphoma 6 (75) Hs.478588 −2 0.0024 ALDH1A3 Aldehydedehydrogenase 1 family, A3 (75) Hs.459538 −1.96 <0.05 NFIL3 Nuclearfactor, interleukin 3 (54) Hs.79334 −1.92 0.0167 regulated CITED2Cbp/p300-interacting transactivator (54) Hs.82071 −1.85 0.0097 SAT1Spermidine acetyltransferase 1 (73, 74, 85) Hs.28491.3 −1.85 0.0063RGC32 RGC32 protein (75) Hs.507866 −1.82 0.0171 G0S2 Putative lymphocyteG0/G1 switch (75) Hs.432132 −1.79 0.0441 gene PTGER2 Prostaglandin Ereceptor 2 (subtype (75) Hs.2090 −1.79 7.87E−06 EP2) PFKFB36-phosphofructo-2-kinase/fructose- (54) Hs.195471 −1.75 0.01562,6-biphosphatase 3 PPAP2B Phosphatidic acid phosphatase, (54) Hs.405156−1.69 0.0028 type 2B IRS2 Insulin receptor substrate 2 (75) Hs.442344−1.67 0.019 ELL2 Elongation factor, RNA (75) Hs.592742 −1.64 0.045polymerase II SLC2A3 Solute carrier family 2, member 3 (54) Hs.419240−1.61 0.0024 PLCB4 Phospholipase C, beta 4 (75) Hs.472101 −1.61 0.0177REV3L REV3-like, catalytic subunit of (75) Hs.232021 −1.61 <0.05 DNApolymerase zeta IL1R1 Interleukin 1 receptor, type I (75) Hs.701982−1.59 0.011 ATP1B1 ATPase, Na+/K+ transporting, (54) Hs.291196 −1.580.0477 beta-1 TGFB2 Transforming growth factor, beta 2 (86, 87)Hs.133379 −1.52 0.0144 Up regulated PENK Proenkephalin (25, 73, 74,Hs.339831 5.08 <0.05 88, 89) SFRP4 Secreted frizzled-related protein 4(73, 74, 89, Hs.658169 4.94 0.0431 90) MMP11 Matrix metalloproteinase 11(25, 73, 74, Hs.143751 4.02 <0.05 89, 91) OLFM1 Olfactomedin 1 (25)Hs.522484 3.45 0.0169 TGFBI Transforming growth factor, beta- (54, 73,74, Hs.369397 3.14 0.0137 induced 89) TK1 Thymidine kinase 1, soluble(73) Hs.515122 2.5 0.0005 MEST Mesoderm specific transcript (75)Hs.270978 2.43 <0.05 homolog THY1 Thy1 cell surface antigen (25, 73)Hs.653181 2.35 0.0231 RRM1 Ribonucleotide reductase M1 (73) Hs.5583932.25 0.0364 polypeptide HMGA2 High-mobility group box 2 (54) Hs.4349532.15 0.002 PGR Progesterone receptor (35, 36) Hs.32405 2.12 0.0322 FBN1Fibrillin 1 (73) Hs.591133 2.03 0.0404 BCL2 B-cell CLL/lymphoma 2 (54)Hs.150749 1.72 0.0321 MARCKS Myristoylated alanine-rich protein (54, 73)Hs.519909 1.52 0.0552 kinase C substrate B. Mid secretory phase (n = 16)Down regulated SCGB2A2 Secretoglobin, family 2A, member 2 (25, 73)Hs.46452 −3.33 0.0447 CYP26A1 Cytochrome P450, subfamily (25, 54)Hs.150595 −2.63 0.0306 XXVIA DEPP/C10orf10 Chromosome 10 open reading(75, 78) Hs.93675 −2.38 0.0129 frame 10 SLC15A2 Solute carrier family15, member 2 (73) Hs.518089 −1.92 0.0107 IGFBP1 Insulin-like growthfactor binding (92) Hs.642938 −1.85 <0.05 protein 1 ATP1B1 ATPase,Na+/K+ transporting, beta-1 (54) Hs.291196 −1.85 <0.05 ELL2 Elongationfactor, RNA (75) Hs.592741 −1.69 0.0461 polymerase II MT1YMetallothionein 1Y (25) Hs.647370 −1.67 <0.05 CAPN6 Calpain 6 (25, 54)Hs.496593 −1.64 <0.05 ENPP1 Ectonucleotide (54) Hs.527295 −1.59 0.0479pyrophosphatase/phosphodiesterase 1 MUC1 Mucin 1, transmembrane (25, 93)Hs.89603 −1.56 0.0391 PIP5K1B Phosphatidylinositol-4-phosphate (54)Hs.534371 −1.56 0.0078 5-kinase, type I, beta FOXO1A Forkhead box O1A(25) Hs.370666 −1.54 0.023 SAT1 Spermidine acetyltransferase 1 (73, 74,85) Hs.28491.3 −1.52 0.0063 Up regulated BCL2 B-cell CLL/lymphoma 2 (54)Hs.150749 1.76 0.0009 PCK1 Phosphoenolpyruvate carboxykinase 1 (54)Hs.1872 1.68 0.0463

Comparison of Moderate/Severe Endometriosis Vs. Normal and Minimal/MildEndometriosis vs. Normal Datasets.

The list of differentially expressed genes during the mid-secretoryphase identified in the current study was compared with the gene listpreviously obtained in a comparison of endometrial gene expressionprofiles during the implantation window in women with vs. withoutminimal/mild endometriosis (Kao L C et al. (2003) Endocrinology144:2870-81). The two datasets shared five up-regulated genes and twelvedown-regulated genes of 1.5 fold or greater (Table 6). Four of the fiveupregulated genes are involved in the immune (GZMA, C4BPA) orinflammatory (S100A8, S100A9) responses.

TABLE 6 Differentially expressed genes in the mid-secretory phaseeutopic endometrium common to both the minimal/mild endometriosis vs.normal and moderate/severe endometriosis vs. normal datasets. Foldchange and p values are those for the current study. Unigene Fold SymbolDescription ID change p value Up-regulated genes (n = 5) S100A8 S100calcium binding protein A8 Hs.416073 2.11 0.0156 GZMA Granzyme AHs.90708 1.87 0.0286 S100A9 S100 calcium binding protein A9 Hs.1124051.71 0.0075 C4BPA Complement component 4 binding protein, Hs.1012 1.70.0018 alpha KIAA0352 KIAA0352 gene product Hs.591025 1.55 3.92E−07Down-regulated genes (n = 12) PTPRR Protein tyrosine phosphatase,receptor type, R Hs.506076 −2.13 0.0015 SLC15A2 Solute carrier family15, member 2 Hs.518089 −1.92 0.0107 PLA2G4A Phospholipase A2, group IVAHs.497200 −1.75 0.0397 RBP4 Retinol binding protein 4 Hs.50223 −1.72<0.05 KIAA1199 KIAA1199 protein Hs.459088 −1.72 0.0349 HLA-DOB Majorhistocompatibility complex, class II, Hs.1802 −1.69 0.001 DOB ANK3Ankyrin 3, node of Ranvier (ankyrin G) Hs.499725 −1.67 0.0202 MUC1 Mucin1, transmembrane Hs.89603 −1.56 0.0391 C11orf8 Chromosome 11 openreading frame 8 Hs.178576 −1.56 0.0194 KRT8 Keratin 8 Hs.533782 −1.560.0345 PIP5K1B Phosphatidylinositol-4-phosphate 5-kinase, Hs.534371−1.56 0.0078 type I S100A1 S100 calcium binding protein Al Hs.515715−1.5 0.0023

TABLE 7 Gene ontology data Gene Ontology Data ES Phase - Enriched GOcategories Biological Processes Up regulated N = 53 total processesMitotic spindle elongation (100) Spindle checkpoint (50) Spindleorganization/biogenesis (50) Traversing start control point (42.8)Mitotic checkpoint (38.5) Deoxyribonucleotide metabolism (25) DNAintegrity checkpoint (25) DNA damage checkpoint (25) Chromosome cycle(23) DNA replication initiation (22.7) G2/M transition of cell cycle(18) Mitosis (16.5) Down regulated Regulation of neurotransmitter lvlsHomeostasis: cell, ion, copper, inorganic cation Actin filament bundleformation Cholesterol biosynthesis Protein-cofactor linkage Transport:organic, carboxylic acid Molecular Functions Up regulated Doublestranded DNA binding Nucleotide binding ATP binding ATPase activityOxidoreductase activity Ribonucleoside diphosphate reductase activitySmall protein activating enzyme activity Ubiquitin like activatingenzyme activity Protein kinase activity Microtubule motor activity Downregulated Cadmium ion binding Copper ion binding Monocarboxylate porteractivity Transport activity: sulfate, organic acid, carboxylic acid,monocarboxylic acid Cellular Components Up regulatedChromosome/chromatin Kinetochore Cytoskeleton Microtubule cytoskeletonSpindle microtubule Microtubule associated complex Kinesin complexIntracellular organelle Nucleus Nuclear lamina Non-membrane boundorganelle Down regulated Cell fraction Membrane fraction Membrane PPhase - Enriched GO categories Biological Processes Up regulated Cellion homeostasis Cation homeostasis Inorganic ion homeostasis Iron ionhomeostasis Metal ion homeostasis Inorganic cation transport Transitionmetal ion transport Ion homeostasis Immune response Humoral immuneresponse Inflammatory response Response to biotic/external stimulusResponse to stress Molecular Functions Up regulated Iron ion bindingCellular Components Up regulated Extracellular region Extracellularspace

Differentially Expressed Genes in the Region of a Locus Showing Linkagewith Endometriosis in a Genome Wide Linkage Analysis.

Recently, Treloar et al published the results of a genome-wide linkageanalysis study involving 1176 families with affected sib pairs (TreloarS A et al. (2005) Am J Hum Genet 77:365-76). This effort identified aregion of significant linkage to endometriosis on chromosome 10q26. Wesearched the genome for genes that fell within the 95% CI of this 10q26locus and compared these against our dataset of differentially expressedgenes in the endometrium of women with endometriosis relative to normalendometrium. This analysis identified the following four genes (foldchange in endometriosis vs. normal endometrium for indicated cyclephase): transforming, acidic coiled-coil containing protein 2, TACC2(10q26; −2.86 ESE, −1.59 MSE), a disintegrin and metalloproteinasedomain 12, ADAM12 (10q26.3; 2.29 ESE), arginyltransferase 1, ATE1(10q26.13; 1.61 PE, 1.57 ESE), and fibronectin type III and ankyrinrepeat domains 1, FANK1 (10q26.2; −1.85 ESE). Other genes of interestnear the 10q26 locus include cytochrome P450, family 26, subfamily A,polypeptide 1, CYP26A1 (10q23-24; −8.33 ESE, -2.63 MSE), retinol bindingprotein 4, RBP4 (10q23-24; −1.72 MSE), pleckstrin and Sec7 domainprotein, PSD (10q24; 2.10 ESE, 1.73 MSE), sorbin and SH3 domaincontaining 1, SH3D5 (10423-24; 1.61 MSE) (Table 8).

TABLE 8 Differentially expressed genes in the vicinity of the locuslinked with endometriosis. Fold Gene Locus Description Unigene ID PhaseChange TACC2 10q26 Transforming, acidic coiled Hs.501252 ES −2.86containing protein 2 MS −1.59 ADAM12 10q26.3 A disintegrin and Hs.655388ES 2.29 metalloproteinase domain 12 ATE1 10q26.13 Arginyltransferase 1Hs.632080 ES 1.57 FANK1 10q26.2 Fibronectin type III and ankyrinHs.352591 ES −1.85 repeat domains 1 HELLS 10q24.2 Helicase,lymphoid-specific Hs.655830 ES 2.99 PSD 10q24 Pleckstrin and Sec7 domainHs.154658 ES 2.1 protein MS 1.73 SORBS1 10q23-24 Sorbin and SH3 domainHs.696027 MS 1.61 containing 1 CYP26A1 10q23-24 Cytochrome P450,subfamily Hs.150595 ES −8.33 XXVIA MS −2.63 RBP4 10q23-24 Retinolbinding protein 4 Hs.50223 MS −1.72 CEP55 10q23.33 Centrosomal protein55 kDa Hs.14559 ES 3.5

Real-Time PCR Validation of Microarray Data.

Three up-regulated and three down-regulated genes in each cycle phasecomparison (of moderate/severe disease vs. women without endometriosis)that showed statistical significance in the microarray dataset werechosen for validation by real-time PCR (FIG. 3). All the genes regulatedin the proliferative phase, as shown by microarray, were confirmed todemonstrate statistically significant regulation in the same directionby real-time PCR (100% concordance). For the early-secretory phase, allgenes selected for validation exemplified similar direction ofregulation to the microarray data, of which 5 were statisticallysignificant for a concordance of 83%. The exception was CYP26A1, whichshowed a fold change in the real-time PCR analysis that did not reachstatistical significance (P=0.097). In the mid-secretory phase, five ofthe six genes selected for validation showed similar directional change,and four of these achieved statistical significance for a concordance of67%. The overall concordance rate of significantly regulated genesbetween the microarray data and the real-time PCR data was 83% (15/18).

Maintenance of a Proliferative Fingerprint in the ESE from Women withEndometriosis.

A striking enrichment of genes involved in mitosis and proliferation inearly secretory endometrium of women with endometriosis was observed(FIG. 4), exceptional insofar as these processes are normallydown-regulated in this phase of the cycle and up-regulated during theproliferative phase (Talbi S et al. (2006) Endocrinology 147:1097-121).The finding of enrichment of genes involved in cell cycle regulation wasconsistent among all ESE specimens from subjects with endometriosis,including those specimens demonstrating concordance for ESE assignmentby both histologic and molecular dating. Although the overall molecularsignature is consistent with the early secretory phase, the genesinvolved in cell proliferation maintain a fingerprint more consistentwith the proliferative phase. A recent study of gene and proteinexpression in murine luminal epithelium provided evidence for directinhibition by P of estrogen-induced DNA synthesis in the cell cycle (PanH et al. (2006) Proc Natl Acad Sci USA 103:14021-6). This study showedprogesterone down-regulated over twenty genes associated with DNAreplication, most notably the minichromosome maintenance (MCM) family.Transcripts for 5 Mcm genes were found to be down-regulated, suggestingthis pathway to be a major target of progesterone action. Interestingly,our study demonstrated up-regulation of all 6 MCM genes in the ESE fromwomen with endometriosis. Other genes associated with cell cycle and DNAreplication that showed down-regulation in response to progesterone inthe study by Pan et al but up-regulation in the ESE from women withendometriosis include PCNA, MKI67, TK1, CCNE1, MAD2L1. Sinceprogesterone is regarded as the key regulator in shifting theendometrium from the proliferative to the differentiated state (GiudiceL C, Ferenczy, A. (1995) In: Adashi E Y et al. (ed) ReproductiveEndocrinology, Surgery and Technology. Raven Press, New York, pp171-194), these findings suggest that the pathway(s) governing thistransition are dysfunctional in the endometrium of subjects withendometriosis.

The molecular mechanisms responsible for the persistence of aproliferative profile in the early secretory endometrium of women withendometriosis are unclear, but could result from altered ligand-receptorinteractions, co-activators/repressors, or post-receptor signaling(Attia G R et al. (2000) J Clin Endocrinol Metab 85:2897-902; BergqvistA, Femo M (1993) Hum Reprod 8:2211-7; Lessey B A et al. (1989) FertilSteril 51:409-15). Differential expression of genes within theprogesterone and epidermal growth factor receptor (EGFR) signalingcascades that may be associated with the maintenance of theproliferative fingerprint was identified in the present study.

The human progesterone receptor (PR) gene contains several biologicallyactive estrogen response elements (Savouret J F et al. (1991) Embo J10:1875-83). Both PR-A and PR-B isoforms are highly expressed inresponse to estrogen in human endometrium before ovulation, but theirexpression is down-regulated by progesterone during endometrialmaturation (Feil P D et al. (1988) Endocrinology 123:2506-13; Lessey B Aet al. (1988) J Clin Endocrinol Metab 67:334-40). In the current study,PR is not suppressed in ESE (fold change 2.12) from women with versuswithout disease. Previously, an immunohistochemical study reportedsignificantly increased PR expression in the epithelial compartment butnot the stromal compartment in the ESE of women with endometriosis(Jones R K et al. (1995) Hum Reprod 10:3272-9. In addition, studies havedemonstrated differential PR isoform expression in the stromal versusepithelial compartments (Mote P A et al. (1999) J Clin Endocrinol Metab84:2963-71).

The FOXO1A gene encodes a progesterone-regulated transcription factorinvolved in cell cycle control and the induction of apoptosis that ismarkedly induced upon decidualization of endometrial stromal cells inboth in vivo and in vitro assays in response to progesterone and cAMP(Accili D, Arden KC (2004) Cell 117:421-6). These data (ESE fold change−2.27, MSE fold change −1.54) corroborate previous findings by others ofreduced FOXO1 gene expression in the endometrium of subjects withendometriosis (Shazand K et al. (2004) Mol Hum Reprod 10:871-7;Matsuzaki S et al. (2005) Fertil Steril 84 Suppl 2:1180-90). Thisfinding was confirmed by real-time PCR (ESE fold change −4.00). Thereduced FOXO1A expression in the endometrium of subjects withendometriosis relative to controls is consistent with a phenotype ofattenuated progesterone response and may play a role in the incompletetransitioning of the endometrium from the proliferative-to-earlysecretory phase.

The molecular mechanism(s) responsible for the persistence of aproliferative profile in the ESE of women with endometriosis may involvenon-steroidal signaling pathways. The present study shows dysregulationof several anti-proliferative genes in the EGFR signaling cascade.Growth factors contribute to maximal proliferation of steroid dependentcells in normal endometrium (Giudice L C (1994) Fertil Steril 61:1-17),and the EGFR pathway is involved in the control of human endometrialgrowth (Irwin J C et al. (1991) Endocrinology 129:2385-92).Mitogen-inducible gene 6 (MIG6) functions as a negative regulator ofEGFR-mediated mitogenic signaling. In the present dataset, MIG6demonstrated statistically significant down-regulation (fold change−2.70) in ESE of subjects with moderate/severe endometriosis relative toendometrium of subjects without disease and this was validated byreal-time PCR (fold change −6.67). Also known as ERFF11 (for ERBBreceptor feedback inhibitor 1), this protein regulates the duration ofMAPK activation via attenuation of EGFR autophosphorylation in a mouseknockout model (Ferby I et al. (2006) Nat Med 12:568-73). Interestingly,the MIG6 locus (1p36.12-33) falls within a region that is a frequentsite of allelic loss in human tumors (Koshikawa K et al. (2004)Hepatogastroenterology 51:186-91; Tseng R C, et al. (2005) Int J Cancer117:241-7), and a recent study using comparative genomic hybridization(CGH) to compare the profiles of eutopic and ectopic endometrium insubjects with endometriosis identified shared allelic loss at 1p36 intwo of three subjects (Wu Y et al. (2006) Gynecol Obstet Invest62:148-159). Down-regulation or loss of MIG6 function may be associatedwith a conferred survival advantage to the refluxed endometrium in theestablishment of endometriotic lesions. Additionally, downregulation ofTOB1 (fold change −2.44) in the ESE of women with endometriosis wasdemonstrated in this study. TOB1, or transducer of ErbB-2, is a cellcycle regulatory protein associated with anti-proliferative activity(Matsuda S et al. (1996) Oncogene 12:705-13). Studies of cultured humanendometrial stromal cells from women with endometriosis demonstratedreduced TOB 1 expression after treatment with IL-1β, a central cytokinein endometriosis (Lebovic D I et al. (2002) Fertil Steril 78:849-54).The TOB1 gene is located on chromosome 17q21, and functional loss ofthis chromosomal region has been observed in endometriotic lesions(Kosugi Y et al. (1999) Am J Obstet Gynecol 180:792-7). The differentialexpression of several genes involved in checking the mitogenic action ofthe EGFR signaling cascade is intriguing.

Dysregulation of Progesterone Target Genes in the Secretory Endometriumof Women with Endometriosis.

In addition to genes involved in cellular proliferation, the secretoryphase profiles of many P-regulated genes in eutopic endometrium of womenwith endometriosis provide further evidence of a relative reduction inprogesterone response. Fifty-four genes in the ESE and sixteen genes inthe MSE evidenced dysregulation in women with disease (Table 5).Metallothioneins (MT) comprise a family of genes clustered on chromosome16q that bind to heavy metal ions and minimize reactive oxygen species.Previous studies demonstrated high MT expression in the secretory phaseendometrium of women without endometriosis (Talbi S et al. (2006)Endocrinology 147:1097-121), and low MT expression in endometrioticimplants (Wicherek L et al. (2005) Gynecol Oncol 99:622-30). In thepresent study, the MTs were among the most highly down-regulated genesin the ESE of women with endometriosis, and this was validated byreal-time PCR (MT1H fold change −33.33). Glutathione peroxidase (GPX3),also up-regulated during the secretory phase in normal endometrium,shares the MT pathway and evidenced significantly reduced expression(ESE fold change −2.78) in the eutopic endometrium of women withendometriosis. The anti-apoptotic gene, BCL-2, is increased in ESE ofwomen with endometriosis, confirming studies by others (Jones R K et al.(1998) Hum Reprod 13:3496-502; Meresman G F et al. (2000) Fertil Steril74:760-6) and suggesting mechanisms for enhanced cell survival in thepathogenesis of this disorder. Interestingly, this gene is negativelyregulated by progesterone in mouse uterus (Jeong J W et al. (2005)Endocrinology 146:3490-505). Another P-regulated gene evidencingstriking dysregulation in the endometrium of subjects with endometriosisis CYP26A1. In normal premenopausal endometrium, the gene expression ofthis retinoic acid catabolic enzyme markedly increases in the secretoryphase (Deng L et al. (2003) J Clin Endocrinol Metab 88:2157-63). In amicroarray study comparing genes induced by progesterone in the uteri ofwild type versus PR knockout mice, CYP26A1 was the most highlyup-regulated gene in response to progesterone (Jeong J W et al. (2005)Endocrinology 146:3490-505). In women with moderate/severe endometriosisrelative to controls, CYP26A1 is among the most significantlydown-regulated genes in both the ESE and MSE, with fold changes of −8.33and −2.63, respectively and validated by real time RT-PCR.Interestingly, the genetic locus for CYP26A1 maps close to a region ofthe genome recently identified to be significantly associated withendometriosis in a genome wide linkage study (Treloar S A et al. (2005)Am J Hum Genet 77:365-76).

Clinical Implications of Attenuated Progesterone Action—ImplantationFailure.

An association between endometriosis and infertility is well established(Hahn D W et al. (1986) Am J Obstet Gynecol 155:1109-13; Schenken R S,Asch R H (1980) Fertil Steril 34:581-7; Steinleitner A et al. (1990)Fertil Steril 53:926-9; Brosens I (2004) Fertil Steril 81:1198-200;Jansen R P (1986) Fertil Steril 46:141-3; Barnhart K et al. (2002)Fertil Steril 77:1148-55). Attenuation of P response at the level of theendometrium may be expected to have a deleterious impact on endometrialreceptivity, and a significant reduction of the implantation rate inwomen with endometriosis undergoing IVF has been reported (Cahill D J,Hull M G (2000) Hum Reprod Update 6:56-66). A prior study identified analtered transcriptome in the endometrium of women with minimum/mildendometriosis during the window of implantation (Kao L C et al. (2003)Endocrinology 144:2870-81). Systematic comparison of the list ofdifferentially expressed genes in the mid-secretory phase of the currentstudy with that of the prior study showed seventeen genes to be common(Table 6). In the context of attenuated P response and implantationfailure, several genes are of interest. MUC-1 and osteopontin, importantin embryo attachment, and glycodelin, important in the immune responseduring implantation, were down-regulated in secretory endometrium ofwomen with versus without endometriosis. We observed a nearly 2-foldreduction in expression of insulin-like growth factor binding protein-1(IGFBP-1) during the window of implantation in the endometrium fromwomen with disease. IGFBP-1 is a sensitive marker for endometrialstromal cell decidualization, and a reduction in IGFBP-1 secretion bycultured endometrial stromal fibroblasts from women with endometriosisrelative to those from women without disease has been documented (KlemmtP A et al. (2006) Fertil Steril 85:564-72). These findings suggestimpaired decidualization of the endometrium in women with endometriosis,which may have important biochemical implications for uterinereceptivity.

Mechanism of Attenuated Progesterone Response.

This study demonstrates abnormalities in eutopic endometrium of womenwith endometriosis, primarily in the early secretory phase, suggestiveof reduced P response in the transition from the proliferative tosecretory phases. In addition, a number of progesterone-regulated genesevidence dysregulation in secretory phase endometrium. In vivoobservations and in vitro studies suggest an intrinsic resistance toprogesterone action in eutopic endometrium of women with endometriosis.

Progesterone resistance exists when normal levels of progesterone elicita subnormal or reduced response. Studies are conflicting regarding thenormalcy of circulating levels of progesterone in women withendometriosis (Brosens I A et al. (1978) Br J Obstet Gynaecol 85:246-50;Cheesman K L et al. (1983) Fertil Steril 40:590-5; Williams C A et al.(1986) Clin Reprod Fertil 4:259-68; Kusuhara K (1992) Am J ObstetGynecol 167:274-7; Cunha-Filho J S et al. (2003) J Assist Reprod Genet20:117-21), and this discrepancy may be secondary to difficulties inboth ascertainment and interpretation of circulating progesteronelevels. A single serum progesterone level may not be representative ofluteal adequacy (Abraham G E et al. (1974) Obstet Gynecol 44:522-5;Laufer N et al. (1982) Am J Obstet Gynecol 143:808-13), and successfulintrauterine pregnancy has been documented with mid-luteal P levels aslow as 3-4 ng/ml (Costello M F et al. (2004) Aust N Z J Obstet Gynaecol44:51-6). Finally, a study of luteal endometrial differentiation inprogrammed cycles of physiologic and subphysiologic exogenousprogesterone replacement in GnRH agonist-suppressed healthy volunteersshowed no differences in endometrial thickness, histology or epithelialintegrin expression at the lower serum progesterone level (Usadi R S etal. (2003) J Soc Gynecol Investig 10:Suppl). This finding supports theargument that the reduced progesterone response in the eutopicendometrium of women with endometriosis is an intrinsic biologicalteration of the endometrium.

The evidence to support progesterone resistance in the setting ofendometriosis is substantial. Endometrial stromal fibroblasts obtainedfrom eutopic endometrium and from ectopic endometrium (endometrioticlesions) demonstrate impaired ability to decidualize in vitro, a findinghighly suggestive of an intrinsic abnormality in theprogesterone-signaling pathway (Klemmt P A et al. (2006) Fertil Steril85:564-72). Others have observed dysregulation of progesterone targetgenes in cultured endometrial stromal cells from women withendometriosis, significant insofar as the progesterone level in theculture medium is well controlled (Bruner-Tran K L et al. (2002) J ClinEndocrinol Metab 87:4782-91). A model for progesterone resistance basedon differential PR isoform expression has been described for ectopicendometrium (Bulun S E et al. (2006) Progesterone resistance inendometriosis: link to failure to metabolize estradiol. Mol CellEndocrinol 248:94-103), and a reduced responsiveness to progesterone ineutopic endometrium has been implicated in disease pathogenesis (OsteenK G et al. (2005) Fertil Steril 83:529-37). The present gene expressionfindings are consistent with resistance to progesterone action in theendometrium of women with endometriosis. The current study provides aframework for further investigation as to the mechanism(s) underlyingattenuated progesterone response in eutopic endometrium of women withendometriosis.

Example 2

This example investigates steroidogenic pathway enzymes in tissue andESFs from women with and without endometriosis.

ESFs were isolated from endometrial biopsies from 15 women with and 7women without endometriosis. After reaching confluence, cells werecultured with 1 μM P₄ (after E₂ 10 nM priming) in a time course of short(3, 6, 48 hours) and long (14 days) term culture, and 0.5 mM of8-bromo-cAMP for 96 hours. IGFBP1 and PRL protein secretion was measuredby ELISA. Alterations in expression of some P₄ and cAMP regulated genes(IGFBP1, PRL, FOXO1A, ERalpha, ERbeta, EBAF, somatostatin (SST), SSTreceptor 2, PRA, PRB) were determined by real-time quantitative(Q)RT-PCR. Purity of ESF populations at passage 2 was evaluated byimmunohistochemistry using cytokeratin, vimentin and CD45 antibodies.Endometrial tissue biopsies were obtained from mid-secretory phaseendometrium from 5 women without and 5 women with severe endometriosis.Regulation of steroidogenic enzymes StAR (UniGene ID Hs.521535), p450scc (UniGene ID Hs.303980), HSD3B1 (, UniGene ID Hs.364941), HSD3B2 (,UniGene ID Hs.654399), Cyp17A1 (UniGene ID Hs.438016), Cyp19A1(UniGeneID Hs.654384), HSD17B1 (HSD17B1 UniGene ID Hs.654385), HSD17B2 (HSD17B2,UniGene ID Hs.162795) by cAMP and/or E₂/P₄ was studied by Q-PCR. (FIG.5)

CyclicAMP was a more potent trigger of decidualization, compared to P₄,as documented by gene and protein expression of decidualization markers.Also, cAMP and P₄ had different effects on decidualization of ESFs fromwomen with vs. without endometriosis suggesting differences inactivation of the cAMP pathway. Steroid hormone biosynthesis is known tobe regulated by cAMP/PKA pathway and starts from activation ofsteroidogenic acute regulatory protein (StAR). StAR regulatescholesterol transport within mitochondria and to P450 scc (side chaincleavage cytochrome, Cyp11A1), which converts cholesterol topregnenolone. In ESFs, cAMP, but not P₄, increased the expression ofboth genes, however, without difference between cells from women withvs. without endometriosis. In tissue samples, StAR, but not P450 scc,was significantly upregulated in endometrium from women withendometriosis compared to those without the disease. 3β-hydroxysteroiddehydrogenase (3βHSD) 1 & 2 catalyse the conversion of pregnenolone toP₄, 17OH to 17-OH—P₄ and DHEA to androstenedione. cAMP significantlyup-regulated mRNAs for both genes in ESFs from women with vs. withoutendometriosis, suggesting decreased conversion of pregnenolone (P₁) toP₄. Down regulation of 3βHSD1, but not 3βHSD2, was observed inmid-secretory endometrial tissue and P₄ stimulated ESFs from women withendometriosis. Cyp17A1 (P450c17) is a cytochrome P450 enzyme thathydroxilates P₁ to P₄, or acts upon 17-OH—P₄ and 17-OHypregnenolone tosplit the side chain off the steroid nucleus. This enzyme was slightlyupregulated by cAMP in ESFs from women with vs without endometriosis,and was slightly down-regulated in endometrial tissue and in ESfsstimulated with P₄. Aromatase (Cyp19A1) functions to aromatize androgensto estrogens (FIG. 6). Aromatase was not regulated by P₄ in ESFs, andwas up-regulated by cAMP in women with endometriosis. In MSE, aromatasewas elevated in women with endometriosis (14.5-fold), but wasundetectable in normal women.

The 17-HSDβ-type 1 (17HSDB1) catalyzes conversion of E₁ to E₂ andtestosterone to androstenedione in liver and endometrium, is regulatedby P₄ and is down-regulated in endometriosis. In the present study,17HSDB1 mRNA was 10-fold up-regulated by cAMP in ESFs from women withvs. without endometriosis. 17HSDB2 in ESCs was up-regulated by cAMP,however, to lesser extent.

This study demonstrates that activation of the PKA pathway stimulates P₄synthesis in ESFs, suggesting regulation of decidualization of thesecells and up-regulation of P₄-responsive genes. ESFs from women withendometriosis demonstrate altered steroidogenic pathway activation, witha decrease in P₄ and an increase in E₂ synthesis. This deficit of P₄ andaccumulation of E₂ may be responsible for the growth and survival ofendometriosis.

TABLE 9 Change in regulation of selected P₄ and cAMP regulated genes inpatients with endometriosis as compared to patients withoutendometriosis. Gene Unigene ID Regulation ESR1 Hs.208124 Up-regulatedESR2 Hs.660607 Up-regulated PGR (PRA) Hs.32405 Up-regulated PGR (PRB)Hs.32405 Up-regulated FOXO1 Hs.370666 Down-regulated PRL Hs.1905Up-regulated IGFBP1 Hs.642938 Down-regulated

Example 3

The aetiology and pathogenesis of endometriosis are not well understood.Hic-5 (Unigene ID 513530), an adaptor-like nuclear receptorco-activator, also known as transforming growth factor beta 1 inducedtranscript 1 and androgen receptor coactivator ARA55, potentiates theactivation of reporter genes by all steroid receptors (GR, AR, MR, andPR), except the ER, and is responsive to progesterone. This exampleshows that dysregulation of Hic-5 is involved in progesterone resistancein endometriosis.

Endometrial biopsies were obtained from mid-secretory phase endometriumfrom women without and with endometriosis: proliferative phaseendometrium (PE) n=3 and n=4 respectively; early secretory endometrium(ESE) n=3 for both groups; mid-secretory endometrium n=5 for bothgroups; late secretory endometrium n=3 and n=4 respectively.Localization of Hic-5 protein was verified by immunofluorescence infull-thickness endometrial biopsies (FIG. 7). Endometrial stromalfibroblasts (ESFs) were isolated from biopsies from 15 women with and 7women without endometriosis. Cells were decidualized with 1 μM P₄ (afterE₂ 10 nM priming) for 14 days, or 0.5 mM of 8-bromo-cAMP for 96 hours.Hic-5, progesterone receptors (PRA and PRB) and Wnt4 gene expression wasanalysed by real-time-RT-PCR.

Immunofluorescence revealed strong expression of Hic-5 in myometriumwithout cyclic variation. In endometrium, Hic-5 was restricted to thestromal compartment, with strong immunoexpression in PE, which decreasedin ESE, and subsequently increased in MSE and towards the end of thecycle. Hic-5 mRNA expression in normal patients replicated the proteinexpression pattern, with gradual decrease from PE to ESE and with asubsequent rise in MSE and LSE (FIG. 8). When levels of Hic-5 mRNA fromwomen with endometriosis were compared to those from women withoutendometriosis, there was a marked down regulation during the P, ESE, andLSE phases (FIG. 9).

Its expression was negatively correlated with PRB and positively withWnt4 mRNA expression throughout the cycle in every patient (r²=−0.687,p≦0.01 and r²=0.618, p≦0.5 respectively), and it correlated positivelywith PRA mRNA expression when patients in each phase were pooled(r²=0.598, p≦0.05). In endometrial biopsies from endometriotic womenHic-5 gene expression was decreased and dysregulated, as were expressionof PRs and Wnt4 in each cycle phase. In culture experiments, ESFsdemonstrated increased Hic-5 gene expression upon decidualization withboth cAMP and E2/P4 stimulation in cells from normal, but notendometriotic women.

This example demonstrates the expression of Hic-5 gene and protein inhuman endometrium, its correlation with PRs, and regulation byprogesterone and cAMP in cultured ESFs. Dysregulation of Hic-5 ineutopic endometrium from women with endometriosis suggests itsinvolvement in the pathogenesis of molecular changes associated withprogesterone resistance in this disorder.

Example 4

Growth of ectopic endometrial tissues in the pelvic cavity, which is ahallmark of endometriosis, is associated with elevated levels ofinflammatory cytokines and increased number of activated macrophages inthe peritoneal environment. While numerous studies have showndifferential gene expression of ectopic endometrium from women withendometriosis, increasing evidence also shows that eutopic endometriumfrom women with endometriosis has dysregulated expression of genes,which are implicated in proliferation of the disease, as well asinfertility and bleeding. In this example, the expression ofinflammatory-associated genes in cultured eutopic endometrial stromalcells from women with versus without endometriosis was examined usingquantitative RT-PCR. Interleukin-8 (IL-8), a cytokine implicated inendometrial cell attachment, invasion, and cell growth, is up-regulated19 fold in eutopic endometrial stromal cells isolated from women withendometriosis compared with cells from women without endometriosis(P=0.03). Interleukin-1 beta and cyclooxygenase-2 mRNA expression weresimilar in cultured endometrial stromal cells from women with vs.without endometriosis. Cell proliferation was similar in eutopicendometrial stromal cells from women with versus without endometriosiswhen cultured in 0%, 2%, or 10% charcoal stripped FBS-containing mediumor in the presence of insulin. Increased IL-8 production inendometriosis is accompanied by a strong increased phosphorylation ofERK ½ (P=0.04), a signaling molecule shown to activate IL-8 expressionin endometrial stromal cells. Progesterone treatment within 60 minsuccessfully diminished the high basal ERK phosphorylation in eutopicendometrial stromal cells from patients with endometriosis, resulting inan amount of ERK phosphorylation similar to the level in cells frompatients without endometriosis. Since phosphorylation of ERK and IL-8expression increases cell migration and invasion of endometrial stromalcells, dysregulation of MAPK activity and IL-8 expression in the eutopicendometrial stromal cells of women with endometriosis may be associatedwith the pathophysiology and spread of the disease.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of diagnosing or providing a prognosis for endometriosis, the method comprising the steps of: (i) obtaining a biological sample from a human subject suspected of or having endometriosis, (ii) detecting in the biological sample altered expression (over or under expression of at least 50% compared to a subject without endometriosis) of a gene or protein, wherein said altered expression is selected from the group consisting of: under expression of monoamine oxidase A (MAOA) gene or protein, under expression of dickkopf homolog 1 (DKK1) gene or protein, under expression of chemokine ligand 13 (CXCL13, B-cell chemoattractant) gene or protein, under expression of metallothionein 1G (MT1G) gene or protein, under expression of metallothionein 1F (MT1F) gene or protein, under expression of secreted phosphoprotein 1 (SPP1, osteopontin) gene or protein, under expression of glutathione peroxidase 3 (GPX3) gene or protein, and under expression of protein tyrosine phosphatase receptor type R (PTPRR) gene or protein, wherein the altered expression correlates with the diagnosis or prognosis for endometriosis, and (iii) providing a diagnosis or prognosis for endometriosis.
 2. The method of claim 1, wherein protein is detected.
 3. The method of claim 1, wherein RNA expression is detected.
 4. The method of claim 1, wherein the sample is an endometrial biopsy.
 5. The method of claim 1, wherein the altered expression is selected from the under expression of the MAOA gene or protein, under expression of dickkopf homolog 1 (DKK1) gene or protein, under expression of chemokine ligand 13 (CXCL13) gene or protein, under expression of metallothionein 1G (MT1G) gene or protein, under expression of metallothionein 1F (MT1F) gene or protein, under expression of secreted phosphoprotein 1 (SPP1) gene or protein, and under expression of glutathione peroxidase 3 (GPX3) gene or protein.
 6. The method of claim 5, wherein the sample comprises early secretory phase endometrial cells or tissue.
 7. The method of claim 1, wherein the altered expression is the under expression of the PTPRR gene or protein and the sample comprises mid secretory phase endometrial cells or tissue.
 8. The method of claim 1, wherein the altered expression is the under expression of the MAOA gene or protein and the sample comprises early secretory phase endometrial cells or tissue.
 9. The method of claim 1, wherein the altered expression is the under expression of the DKK1 gene or protein and the sample comprises early secretory phase endometrial cells or tissue.
 10. The method of claim 1, wherein the altered expression is under expression of the CXCL13 gene or protein and the sample comprises early secretory phase endometrial cells or tissue.
 11. The method of claim 1, wherein the altered expression is under expression of the MT1G gene or protein and the sample comprises early secretory phase endometrial cells or tissue.
 12. The method of claim 1, wherein the altered expression is under expression of the MT1F gene or protein and the sample comprises early secretory phase endometrial cells or tissue.
 13. The method of claim 1, wherein the altered expression is under expression of the SPP1 gene or protein and the sample comprises early secretory phase endometrial cells or tissue.
 14. The method of claim 1, wherein the altered expression is under expression of the GPX3 gene or protein and the sample comprises early secretory phase endometrial cells or tissue.
 15. A method of diagnosing or providing a prognosis for endometriosis, the method comprising the step of: (i) detecting, in a biological sample from a human subject suspected of or having endometriosis, altered expression (over or under expression) of a gene or protein wherein said altered expression is selected from the group consisting of: under expression of monoamine oxidase A (MAOA) gene or protein, under expression of dickkopf homolog 1 (DKK1) gene or protein, under expression of chemokine ligand 13 (CXCL13, B-cell chemoattractant) gene or protein, under expression of metallothionein 1G (MT1G) gene or protein, under expression of metallothionein 1F (MT1F) gene or protein, under expression of secreted phosphoprotein 1 (SPP1, osteopontin) gene or protein, under expression of glutathione peroxidase 3 (GPX3) gene or protein, and under expression of protein tyrosine phosphatase receptor type R (PTPRR) gene or protein, wherein over or under expression of at least 50% compared to a human subject without endometriosis provides a diagnosis or prognosis for endometriosis. 